# Patent

## Title
Protofibril selective antibodies and the use thereof

## Abstract

The invention relates to an isolated antibody, or fragment thereof, having high affinity for human Aβ protofibrils. The invention further relates to compositions that include the antibody, or a fragment thereof, and a pharmaceutically acceptable buffer. The invention further relates to a method of preventing or treating Alzheimer's disease, which includes the step of administering to a patient having or suspected of having Alzheimer's disease such an antibody, or fragment thereof or a composition that includes the antibody or a fragment thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 12/294,207, filed Sep. 23, 2008, which is the national stage of International Application No. PCT/SE2007/000292, filed Mar. 23, 2007, which claims the benefit of SE 0600662-1, filed Mar. 23, 2006, and SE 0602591-0, filed Nov. 30, 2006.


FIELD OF INVENTION
This invention pertains to the prevention, treatment and diagnosis of neurodegenerative diseases, in particular Alzheimer's disease, and other similar disease. More precisely, to high affinity 10−7M, preferably 10−8M, even less than 10−9M or less than 10−10 M or 10−11 M antibodies, selective for amyloid beta protein (Aβ) in its protofibril conformation and of IgG class and IgG1 or IgG4 subclass or combinations thereof or mutations thereof, retaining high Fc receptor binding and low C1 (C1q) binding, effective in clearance of Aβ protofibrils and with reduce risk of inflammation.
BACKGROUND
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder causing cognitive, memory and behavioural impairments. It is the most common cause of dementia in the elderly population affecting roughly 5% of the population above 65 years and 20% above 80 years of age. AD is characterized by an insidious onset and progressive deterioration in multiple cognitive functions. The neuropathology involves both extracellular and intracellular argyrophillic proteineous deposits. The extracellular deposits, referred to as neuritic plaques, mainly consist of amyloid beta protein (Aβ) surrounded by dystrophic neurites (swollen, distorted neuronal processes). Aβ within these extracellular deposits are fibrillar in its character with a β-pleated sheet structure. Aβ in these deposits can be stained with certain dyes, e.g. Congo Red, and display a fibrillar ultra structure. These characteristics, adopted by Aβ in its fibrillar structure in neuritic plaques, are the definition of the generic term amyloid. The classic intracellular AD pathologic lesion is the neurofibrillary tangle (NFT) which consists of filamentous structures called paired helical filaments (PHFs), composed of twisted strands of hyperphosphorylated microtubule-associated protein tau. Frequent neuritic plaques and neurofibrillary tangle deposits in the brain are diagnostic criteria for AD, as carried out post mortem. AD brains also display macroscopic brain atrophy, nerve cell loss, local inflammation (microgliosis and astrocytosis) and often cerebral amyloid angiopathy (CAA) in cerebral vessel walls.
Two forms of Aβ peptides, Aβ40 and Aβ42, are the dominant species in AD neuritic plaques while Aβ40 is the prominent species in cerebrovascular amyloid associated with AD. Enzymatic activities allow Aβ to be continuously formed from a larger protein called the amyloid precursor protein (APP) in both healthy and AD afflicted subjects in all cells of the body. Two major APP processing events through β- and γ-secretase activities enables Aβ production, while a third enzyme called α-secretase, prevents Aβ generation by cleavage inside the Aβ sequence (Selkoe, 1994; Ester 2001;U.S. Pat. No. 5,604,102). The Aβ42 is a fortytwo amino acid long peptide, i.e. two amino acids longer at the C-terminus, as compared to Aβ40. Aβ42 is more hydrophobic, and does more easily aggregate into larger structures of Aβ peptides (Jarret 1993) such as Aβ dimers, Aβ trimers, Aβ tetramers, Aβ oligomers, Aβ protofibrils or Aβ fibrils. Aβ fibrils are hydrophobic and insoluble, while the other structures are all less hydrophobic and soluble. All these higher molecular structures of Aβ peptides are individually defined based on their biophysical and structural appearance e.g. in electron microscopy, and their biochemical characteristics e.g. by analysis with size-exclusion chromatography/western blot. These Aβ peptides, particularly Aβ42, will gradually assemble into a various higher molecular structures of Aβ during the life span. AD, which is a strongly age-dependent disorder, will occur earlier in life if this assembly process occurs more rapidly. This is the core of the “amyloid cascade hypothesis” of AD which claims that APP processing, the Aβ42 levels and their assembly into higher molecular structures is a central cause of AD. All other neuropathology of AD brain and the symptoms of AD such as dementia are somehow caused by Aβ or assembled forms thereof.
Aβ can exist in different lengths i.e. 1-39, 1-40, 1-42 and 1-43 and fragments sizes i.e. 1-28 and 25-35. Truncations might occur at the N-terminus of the peptide. All these peptides can aggregate and form soluble intermediates and insoluble fibrils, each molecular form having a unique structural conformation and biophysical property. Monomeric Aβ1-42 for example, is a 42 amino acid long soluble and non toxic peptide, that is suggested to be involved in normal synapse functions. Under certain conditions, the Aβ1-42 can aggregate into dimers, trimers, tetramers, pentamers up to 12-mer and higher oligomeric forms, all with its distinct physicochemical property such as molecular size, EM structure and AFM (atomic force microscopy) molecular shape. An example of a higher molecular weight soluble oligomeric Aβ form is the protofibril (Walsh 1997), which has an apparent molecular weight >100 kDa and a curvelinear structure of 4-11 nm in diameter and <200 nm in length. It has recently been demonstrated that soluble oligomeric Aβ peptides such as Aβ protofibrils impair long-term potentiation (LTP) a measure of synaptic plasticity that is thought to reflect memory formation in the hippocampus (Walsh 2002). Furthermore, oligomeric Arctic Aβ peptides display much more profound inhibitory effect than wtAβ on LTP in the brain, likely due to their strong propensity to form Aβ protofibrils (Klyubin 2003).
There are also other soluble oligomeric forms described in the literature that are distinctly different from protofibrils. One such oligomeric form is ADDL (Amyloid Derived Diffusible Ligand) (Lambert 1998). AFM analysis of ADDL revealed predominantly small globular species of 4.7-6.2 nm along the z-axis with molecular weights of 17-42 kDa (Stine 1996). Another form is called ASPD (Amyloidspheroids) (Hoshi 2003). ASPD are spherical oligomers of Aβ1-40. Toxicity studies showed that spherical ASPD >10 nm were more toxic than lower molecular forms (Hoshi 2003). This idea has gained support from recent discovery of the Arctic (E693) APP mutation, which causes early-onset AD (US 2002/0162129 A1; Nilsberth et al., 2001). The mutation is located inside the Aβ peptide sequence. Mutation carriers will thereby generate variants of Aβ peptides e.g. Arctic Aβ40 and Arctic Aβ42. Both Arctic Aβ40 and Arctic Aβ42 will much more easily assemble into higher molecular structures i.e. protofibrils. Thus, the pathogenic mechanism of the Arctic mutation suggests that the soluble higher molecular protofibrils are causing AD and contains a specific unique epitope i.e. “the AD disease epitope”.
In the Alzheimer's disease (AD) brain, extracellular amyloid plaques are typically found in parenchyma and vessel walls. The plaques are composed of amyloid (Aβ38-43 amino acid long hydrophobic and self-aggregating peptides, which gradually polymerize prior to plaque deposition. The soluble AD oligomeric species have been proposed to be better disease correlates than the amyloid plaques themselves (McLean et al., 1999; Näslund et al., 2000). Among these pre-fibrillar intermediate Aβ species, oligomeric forms have been shown to elicit adverse biological effects both in vitro and in vivo (Walsh et al., 2002) and may thus play a central role in disease pathogenesis. Several oligomeric Aβ species of various molecular sizes are known. Importantly, the conformation of monomeric, oligomeric and fibrillar forms of Aβ are different and can be targeted by conformational selective antibodies. The identity of the main Aβ pathogen is unclear, although some evidence suggests high-molecular weight Aβ oligomers to be especially neurotoxic (Hoshi et al., 2003).
Pathogenic mutations in the amyloid precursor protein (APP) gene, causing early onset AD have been described. One of them, the Swedish APP mutation (Mullan et al., 1992), causes increased levels of Aβ. The other the Arctic APP mutation (E693G) located within the Aβ domain, was found to enhance the formation of protofibrils, large Aβ oligomers, suggesting these Aβ intermediates to be particularly pathogenic ((US 2002/0162129 A1; Nilsberth et al., 2001). The identification of the Arctic APP mutation and the elucidation of toxic effects for Aβ protofibrils have increased the focus on Aβ oligomers in AD pathogenesis.
Active immunization as a therapeutic strategy for Alzheimer's disease was first reported by (Schenk et al. 1999). The target for the immunization strategy was the fibrillar form of Aβ found in Alzheimer plaques. A recent clinical phase I/II trial of active Aβ vaccination using fibrillized Aβ as a vaccine (AN-1792) had to be halted because of the development of meningoencephalitis in a small number of patients (Bayer et al., 2005). The side effects seen in this study were likely caused by anti-Aβ antibodies reacting against fibrillar amyloid in vessel walls. The fibrillary amyloid in CAA is in close proximity to the blood-brain-barrier (BBB) and the antigen-antibody reaction could thus generate damage to the BBB leading to infiltration of T-lymphocytes into the CNS (Pfeifer et al., 2002; Racke et al., 2005). Moreover, only a minority of the participating patients displayed an immune response to the Aβ vaccine. Although the study ended prematurely, it seems to imply that active Aβ immunization may be beneficial only to a subset of AD patients.
Monoclonal antibodies selective for human Aβ protofibrils have been described (US 2002/0162129 A1). The method to generate highly pure and stable human Aβ protofibrils, involves the use synthetic Aβ42 peptides with the Arctic mutation (Glu22Gly). The mutation facilities immunization and hybridoma screening for Aβ protofibril selective antibodies. Importantly, these antibodies bind both wild-type Aβ protofibrils and Aβ-Arc protofibrils (PCT/SE 2005/000993).
Antibodies that are selective towards other conformations of Aβ such as Aβ fibrils (O'Nuallain 2002), micellar Aβ (Kayed 2003), ADDL (Lambert 2001), have been described. However, non of these are Aβ protofibril selective.
SUMMARY OF TILE INVENTION
The present invention pertains to improved antibodies i.e. high affinity (less than 10−7 M) Aβ protofibril selective antibodies of class IgG and subclass IgG1 or IgG4 or combination thereof or mutations thereof, with reduced risk of inflammation, for improved prevention, treatment and diagnosis of Alzheimer's disease, Downs syndrome or other neurodegenerative disorders. Said antibodies have been developed by classical hybridoma techniques and antibody engineering.
The invention discloses the consensus amino acid sequence of the CDR1-3 regions on the VL and VH chains from antibodies that selectively bind oligomeric Aβ forms, i.e. Aβ protofibrils constituting the “Alzheimer disease epitope”, combined with modifications of the Fc region to reduce complement factor C1q binding, reducing the risk for complement activation and inflammation.
The constant region of an antibody has many important functions notably binding Fc-receptors and complement factor C1q. The latter function has been inactivated to avoid inflammatory reactions.
In summary, this type of high affinity protofibril selective antibodies have the following distinct advantages as compared to other known immunotherapeutic treatment modalities:

    1) targets disease causing Aβ protofibrils with high affinity
    2) reduces the risk for inflammatory side-effects i.e. meningioencephalitis, by low or no binding to complement factor C1q
    3) high affinity antibody reduces the clinical dose needed for an effective treatment
    4) provides a modality of accurate dosing
    5) less binding to Aβ fibrils in the blood vessel wall i.e. CAA, reducing the risk for inflammatory side-effects.
    6) Less antibody is bound in the periphery, thus more will cross the blood brain barrier and be available for binding and elimination of Aβ oligomeric forms in the brain.


One aspect of the invention is the discovery of the antibody consensus amino acid sequence of the CDR regions that bind human wild type Aβ protofibrils (Example 1). This discovery defines the binding sites (CDR regions) that confer high affinity and high selectivity for wild-type human Aβ protofibrils for use as therapeutics or diagnostics. The basic structure of an immunoglobulin (IgG) molecule comprises two identical light chains and two identical heavy chains linked together by disulphide bridges (FIG. 1). The light chain, which is either lambda or kappa, has a variable region (VL) and a constant region (CL) of approximately 110 amino acid residues each. The heavy chain has a variable region (VH) of about 110 amino acid residues, but a much larger constant region (CH) of 300-400 amino acid residues, comprising CHγ1, CHγ 2 and CHγ3 regions or domains.
The constant region (Fc) activates the complement system and binds to a Fc receptor on macrophages, microglia and neutrophiles, which ingest and destroys infecting microorganisms or foreign/non-self antigens. This function is particular important since it is part of the therapeutic principle of the antibody, i.e. Fc receptor mediated microglial phagocytosis and clearance of Aβ protofibrils. Other antibody mediated clearance mechanisms are also operating, i.e. anti-aggregation properties of Aβ antibodies and clearance of Aβ protofibrils in the periphery, according to the sink hypothesis. The variable region of the heavy and light chains contains 3 hyper variable regions called complementary determining regions or CDRs. The CDR regions are short stretches of about β-23 amino acid long, located in the VL and VH regions. The six CDRs regions on one “arm” of the antibody forms the “pocket” that binds the antigen. FIG. 1 shows the basic structure of an IgG immunoglobulin and its subdomains.
Another aspect of the invention pertains to protofibril selective antibodies of high affinity. Affinities in the range of 10−7M preferably 10−8 M, even less than 10−9M, less than 1010 M, or less than 10−11M for protofibrils are described (Example 2). These antibodies have the advantage that they can be administered at lower doses compared to antibodies with affinities in the 10−6 M range. This has significant clinical advantage in that these high affinity antibodies, which are administered by injection, can be given subcutaneously since only a low amount of the antibody is needed to achieve efficacy. Administration modalities are not limited to subcutaneous injections. Furthermore, the lower doses needed for efficacy will reduce cost of goods for production of the antibody.
Another aspect of the invention is that the antibodies are of IgG class, suitable for therapeutic use since it can pass over the blood brain barrier. Clearance of Aβ protofibrils in the brain parenchyma is achieved by Fc receptor mediated phagocytosis by microglia cells. Other anti-Aβ clearance mechanisms are likely to operate as well. This clearance of soluble Aβ protofibrils is a central mechanism of the treatment. Aβ protofibrils are considered highly neurotoxic, initiating and driving the disease process. Clearance of Aβ protofibrils in the brain is of significant clinical value. In addition to clearance of Aβ protofibrils, other Aβ oligomeric forms including Aβ fibrils, will be reduced indirectly via removal of Aβ protofibrils since different Aβ aggregated forms, i.e. dimers, trimers, tetramers and higher oligomeric forms including protofibrils and fibrils, are in equilibrium. Example of reduction of plaques, which contain Aβ fibrils, is shown in a Alzheimer transgenic mouse model (APPswe) after 72 hour treatment with a high affinity protofibril selective antibody (mAb 158) (Example 3). Hence, clearance of Aβ protofibrils by said antibody will also have the advantage to indirectly reduce other Aβ aggregated or oligomeric forms.
Yet another aspect of the invention is a high affinity human Aβ protofibril selective antibody of subclass IgG1, which has a high affinity for human FcγRI receptors present on microglial cells in the brain. A high affinity antibody will lead to efficient clearance of Aβ protofibrils which will be of significant therapeutic value. Hence, the antibodies will exhibit clearance of Aβ protofibrils, both in CNS and periphery as compared to other immunotherapeutic strategies such as active vaccination or monoclonal antibody treatments with other monoclonal antibodies of IgG1 subclass targeting other Aβ forms. Importantly, the treatment will be efficient early in the disease process when toxic soluble Aβ spices such as Aβ protofibrils are present at elevated levels but also later in the disease process. Elevated levels of oligomeric Aβ forms have been described in a transgenic mouse model exhibiting the Swedish and Arctic mutations APP swearc (Lord A. et al. 2006). Yet another aspect of the invention is that the high affinity Aβ protofibril selective antibodies can reduce or inhibit Aβ aggregation thereby reducing levels of soluble oligomeric Aβ forms in the brain.
Yet, another aspect of the invention is that the high affinity Aβ protofibril selective antibodies can bind oligomeric forms of Aβ, i.e. Aβ protofibrils outside CNS as well, thereby shifting the equilibrium of said Aβ forms over the blood brain barrier in such a way as to lower CNS levels of said Aβ forms (drainage).
As discussed above, the Elan clinical study using an Aβ vaccine (AN-1792) selective for Aβ fibrils to treat Alzheimer patients resulted in a side-effect, i.e. meningioencephalitis, in 6% of the cases. The strategy to target Aβ fibrils, that are the core of amyloid plaques present in the brain parenchyma but importantly also in the blood vessel walls, resulted in severe side-effects. The side-effects was most likely caused by the binding of the antibodies to CAA (Cerebral Amyloid Angiopathy) in the blood vessel walls of the brain, starting an inflammatory process. This significant clinical problem is avoided by the improved high affinity protofibril selective antibodies with reduced complement activation activity. These antibodies will retain high clearance efficacy of Aβ protofibrils reduced risk of side-effects, i.e. meningioencephalitis.
Another aspect of the invention is that the high affinity protofibril selective antibodies have low Aβ fibril binding (See example 2), reducing the risk for side effects, by less binding to Aβ fibrils present in CAA.
Yet another aspect of the invention is that the high affinity Aβ protofibril selective IgG antibodies are engineered to reduce complement factor C1q binding to the CH2 domain of IgG1 and reduce complement activation and risk of inflammation. This modification can be done in several different ways. One way is to make a chimeric antibody where the CHγ2 domain of the IgG1 constant region has been deleted and exchanged for the corresponding domain from IgG4 or part of the domain that confers C1q binding. It is well established that IgG4 does not bind C1q and hence does not activate the complement cascade. To achieve this the constant region of the heavy chain (CH) is engineered is such a way as to combine the high affinity Fc-receptor domain (CHγ3) on IgG1 with the IgG4 domain (CHγ2) which has no binding for the complement factor C1q. This new antibody containing the chimeric constant heavy chain (IgG1:CHγ1, CHγ2:IgG4, CHγ3:IgG1) will have the important properties of both efficient clearance of Aβ protofibrils through Fc-receptor mediated phagocytosis and reduced risk for side-effects, i.e inflammation such as meningioencephalitis.
Yet another way of reducing the risk of inflammation is to alter the oligosaccharides structure of the antibody which will reduce complement factor C1q binding and complement activation. 30 different structures of the complex biantennary oligosaccharides at Asn-297 in human IgG1 has been described. The absence of CH2 associated carbohydrates is believed to cause a conformational change in the “hinge” region of the antibody, reducing interaction efficacies with effector molecules and loss of complement activation function and C1q binding.
The modification of a high affinity human Aβ protofibril selective antibody by site-directed mutagenesis of Asn-297 to any other amino acid will generate an antibody of retained Fc-receptor binding with less C1q binding and hence reduced risk of inflammation in particular at the blood brain barrier. An alternative to modify the glycosylation on the antibody is to expressing the antibody in a cell type where the enzyme N-acteylglucosaminyl-transferase I has been inactivated. This will yield an antibody with altered carbohydrate structure at Asn-297. A structure of Man5GlcNAc2, but not limited to this structure, is formed. This carbohydrate modification will reduce complement factor C1q binding and inhibit inflammation (Wright at al. 1998). Alternatively, glycosylated protofibril selective antibodies can be achieved by culturing cells expressing antibodies in the presence of tunicamycin, which inhibits glycosylation. These antibodies will have altered complement activating activity as well as altered Fc-receptor function (Leatherbarrow et al. 1985). Screening of clones expressing antibodies with low complement activation and high Fc-receptor binding will generate protofibril selective antibodies that exhibit high Fc-mediated clearance of Aβ protofibrils and low C1q binding.
Yet another aspect of the invention is a high affinity human Aβ protofibril selective antibody, of IgG1 subclass, where the complement factor C1q binding site has been modified, i.e. Pro331>Ser331 (Xu et al. 1994), in such a way as to reduce or inhibit binding of complement factor C1q, for the treatment or prevention of AD. The proline residue at position 331 in human IgG1 can also be changed to a threonine or glycine or any other polar amino acid. This modification can be achieved by standard molecular biology techniques such as site-directed mutagenesis or DNA deletions.
Yet another aspect of the invention is the use of high affinity human Aβ protofibril selective IgG antibodies to specifically determine protofibril levels in human tissues, in particular in cerebrospinal fluid, blood, urine or saliva as a diagnostic tool or biomarker for Alzheimer's disease. Levels of human Aβ protofibrils in CSF or blood are likely to be different as compared to a matched elderly control group not having Alzheimer's disease. A person who is developing Alzheimer's disease is likely to have increased levels of Aβ protofibril levels in CSF or blood. Hence, by determination of Aβ protofibril levels in CSF or blood an early diagnosis of the disease can be made. This is possible to achieve with the new high affinity Aβ protofibril selective antibodies in combination with a sandwich ELISA method (Example 2A), where Aβ protofibrils have been determined down to 10 pM level. Interference of other Aβ forms such as Aβ fibrils, Aβ monomers and Aβ fragments (1-16; 17-40) in the assay, is 10% or less.
The invention further pertains to the use of a high affinity protofibril specific antibodies for determinations of Aβ protofibrils in human and animal tissues, for example, cerebrospinal fluid, blood, serum, urine and brain tissue but not limited to these tissues, providing for a possible diagnostic method for Alzheimer's disease. Suitable methods for assaying Aβ protofibrils in these tissues as well as in cell cultures using an anti-Aβ protofibril antibody are immunoassays such as ELISA, RIA, Western blotting or dot blotting. The method would be suitable to follow treatment efficacy (protofibril reduction) in clinical trials and suitable as a diagnostic test for Alzheimer's disease or Down's syndrome.
Since Aβ protofibrils levels are very low in CSF and blood, a high affinity Aβ protofibril selective antibody is needed in a diagnostic test based on an ELISA method, to be able to measure low levels of Aβ protofibrils. Other supersensitive methods such as proximity ligation (Example 4) (Gullberg 2004) or similar amplification systems or Biacore or similar techniques, can be used to increase sensitivity. The proximity ligation technique is based on the discovery that different antibodies, raised against different epitopes on an analyte (in this case a protein), may bind near each other on said analyte. If said different antibodies are conjugated to oligonucleotides, the distance between said oligonucleotides will be short enough for a connector oligonucleotide, with the aid of ligation components, to form a bridge between the oligonucleotides. Amplification components are also added, upon which RT-PCR may be performed. By this principle, an amplifiable DNA sequence, reflecting the identity and amount of the target protein, is generated. This technique makes it possible to obtain an enhanced signal response and thus to detect lower concentrations of analyte.
The present inventors surprisingly discovered that a modified proximity ligation technique may also be used with their Aβ protofibril-specific antibodies, to detect low concentrations of larger Aβ peptide structures, i.e. Aβ protofibrils but not Aβ monomers. They discovered that the Aβ peptides, in the protofibril conformation, exhibits a structure (repetitive units) that makes it possible for two antibodies, according to the present invention, to bind sufficiently near each other on the protofibril. If said antibodies are conjugated to oligonucleotides, said oligonucleotides may be bridged using a connector oligonucleotide. PCR is performed using amplification components. By this principle, an amplifiable DNA sequence, reflecting the identity and amount of the target protofibril, is generated (see FIG. 4A).
Proximity ligation or a version of the technique called “rolling circle”, is a highly sensitive technique and particularly well suited for detection of polymeric structures with repeated sequences, such as Aβ protofibrils to be used for diagnosis of Alzheimer's disease and other neurodegenerative disorders.
The invention further pertains to the use of high affinity protofibril specific antibodies in imaging for detection, localization and quantitation of Aβ protofibrils in human and animal tissues. The antibody could be label with a radioactive ligand such as I131, C14, H3 or Gallium68, but not limited to these radioisotopes, for detection purposes. The method will be suitable as a diagnostic tool for Alzheimer's disease or Down's syndrome.
Yet another aspect of the invention is to make the antibody spices specific for use in veterinary medicine. The diagnostic methods outlined are also suitable for veterinary use.
Another aspect of the invention is the humanization of said antibodies to avoid side-effect, i.e. to avoid an immunoresponse against said antibodies in humans when used as a therapeutic or diagnostic agent.
Yet another aspect is a formulation of the antibody in a physiological buffer, for example PBS but not limited to PBS, suitable for administration to humans and animals. The antibody product can be freeze dried for better stability. The freeze dried formulation can contain an excipient such as manitol but not limited to manitol to stabilize the product after freeze drying.
The antibody product can contain an antibacterial agent.
The antibodies or fragments according to the inventions may exhibit amino acid deletions, substitutions and insertions within said CDR regions and/or its framework. Inserted or substituted amino acids may also be amino acid derivatives, with the proviso that the affinity and specificity of the antibody is still intact.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the basic structure of an immunoglobulin molecule.
FIG. 2 shows characterization of a high affinity protofibril selective monoclonal antibody using a sandwich ELISA (A) and a competitive ELISA (B).
FIG. 3 shows the therapeutic efficacy of a high affinity protofibril selective antibody in a transgenic mouse model.
FIG. 4 shows human Aβ protofibrils measured at pM levels by the proximity ligation technique.
FIG. 5 shows that mAb158 does not exhibit reactivity with any amyloid other than the Aβ fibril (A), positive controls (B), and Western blots with the 6E10 antibody (C).
FIG. 6 shows assays measuring Aβ protofibrils.
FIG. 7 shows the Aβ protofibril concentration in HEK-cell culture media (A) and in mouse brain homogenates (B).
FIG. 8 shows the Aβ protofibril levels in APPSwearc transgenic mouse brain TBS extracts after 4 months treatment with either mAb 158 or placebo.
FIG. 9 shows the total Aβ levels in APPSwearc transgenic mouse brain formic acid extracts after 4 months treatment with either mAb158 or placebo.
FIG. 10 shows the pKN100 vector.
FIG. 11 shows the pG1D200 vector.
FIG. 12 shows Aβ monomer binding by chimeric and mouse 158 antibodies.
FIG. 13 shows competition of monomeric or protofibrillar Aβ for binding to chimeric 158 or mouse 158 antibody.
FIG. 14 shows competition of monomeric or protofibrillar Aβ for binding to chimeric 158 or mouse 158 antibody.
FIG. 15 shows the SignalP algorithm 6 result for the K5.1# leader sequence (SEQ ID NO:103). The SignalP algorithm 6 generates the combination score Y from the cleavage site score C and the signal peptide score S.
FIG. 16 shows the SignalP algorithm 6 result for the NL-1 HV leader sequence (SEQ ID NO:112). The SignalP algorithm 6 generates the combination score Y from the cleavage site score C and the signal peptide score S.
FIG. 17 shows the M99649 signal peptide (SEQ ID NO:153) cutting prediction. The SignalP algorithm 10 generates the combination score Y from the cleavage site score C and the signal peptide score S.
FIG. 18 shows the A19 signal peptide (SEQ ID NO:239) cutting prediction. The SignalP algorithm 10 generates the combination score Y from the cleavage site score C and the signal peptide score S.



EXAMPLES
The following examples are provided for illustration and are not intended to limit the invention to these specific examples.
Example 1
Human wild-type Aβ protofibril selective monoclonal antibodies were cloned and sequenced. The amino acid sequence of the variable heavy chain region (VH) and the variable light chain region (VL) are shown in Table 1. The positions of the CDR regions 1-3 are underlined and shown as well in Table 2 and 3. The amino acid sequences of the CDR regions form the structural basis for binding human wild-type Aβ protofibrils constituting the “Alzheimer disease epitope”.
The amino acid sequence of the CDR regions 1-3 of the VL and VH chains for a high affinity protofibril specific antibody BA9/158 is shown in Table 1, 2 and 3. Sequencing data of other protofibril selective antibodies (BA2, BA3, BA4 and BA7) provide alternative amino acids sequences of the CDR regions but not limited to these. The combined amino acid sequences of the CDR1-3 regions of the VH and VL chains create the molecular “pocket” which binds human Aβ wild-type protofibrils with high affinity and specificity. This “pocket” forms the structural basis of the “Alzheimer's disease epitope”. Variations in the CDR amino acid sequence length are observed in both the VH chain and the VL is compatible binding to human Aβ protofibrils (Table 2 and 3). A shorter CDR region provides a more restricted three dimensional structure of the binding pocket of the antibody, whereas a longer is more flexible.
We claim the CDR sequences as shown in Tables 1, 2 and 3 as well as amino acid sequences in the “mouse framework” regions of the VH and VL chains, i.e. outside the CDR regions as well as the human VL and VH framework regions for protofibril specific antibodies as shown in Table 4 and 5, but not limited to those.
The amino acid sequence of the framework region of VL and VH regions 1-3 of the VL and VH chains from a high affinity protofibril specific antibody BA9/158 is shown in Table 4 and 5.
Other amino acid substitution in the CDR regions than what is shown in Table 1, 2 and 3 are compatible with high affinity and high specificity binding to human wild-type Aβ protofibrils. Where a polar amino acid is present in a particular position in a CDR region that particular amino acid can be substituted by another polar amino acid, with retained or improved high affinity and specificity binding to Aβ protofibrils. Likewise, if a non-polar or negatively or positively charged amino acids is present at a certain position, that amino acid can be substituted for by a similar amino acid from the same group.
Also, a particular amino acid or amino acids are exchanged in any position in the CDR regions by functional equivalents that confers a similar function and structure to the antibody.
Example 2
Characterization of an High-Affinity Human Aβ Wild-Type Profibril Selective Monoclonal Antibody by ELISA
Example 2 shows a high affinity protofibril selective antibody that cross-reacts a 200-1000-fold less with Aβ monomers and less than 40-fold with Aβ fibrils, as measured by a sandwich ELISA (FIG. 2A). From competitive ELISA experiments, the antibody has a strong affinity for human Aβ42 wild-type protofibrils, but only very weak affinity for the N-terminal part of the Aβ peptide and Aβ monomers. No binding was observed to the C-terminal fragment of Aβ (FIG. 2B). Furthermore, the antibody does not cross-react with other types of amyloids, like medin or transthyretin. Furthermore the antibody does not recognize human APP, the abundant precursor of Aβ.
In FIG. 2A a sandwich ELISA is shown. Antibody 158 was coated in the wells and different Aβ forms subsequently added to the well in increasing concentrations.
Measurement of bound Aβ forms was made by adding biotinylated mAb 158 and HRP labelled Streptavidine. Colour development was measured according to the procedure recommended by the manufacturer.
In FIG. 2B a competitive ELISA is shown. An ELISA plate was coated with human Aβ protofibrils. Antibody 158 was subsequently incubated with increasing amounts of different Aβ forms (competition). The incubation mix was added to the microtiter plate wells and free antibody was allowed to bind to immobilized protofibrils in the wells. Bound 158 antibody was measured by a second antibody using standard procedures.
Example 3
The efficacy of high affinity Aβ protofibril selective antibody was determined in an Alzheimer transgenic mouse model (APPswe) by an acute intracranial injection. Transgenic mice used for efficacy evaluation express human APP, with the Swedish mutation (APPSwe). In this paradigm, antibodies are injected directly into plaque-rich regions of the brain parenchyma and effects on neuropathology are assessed after 72 hours (Wilcock et al., 2003). Other studies have shown that the direct application of anti-Aβ antibodies results in a rapid clearance of amyloid deposits in vivo (Bacskai et al., 2001; Brendza et al., 2005). The injection of high affinity Aβ protofibril selective antibody leads to a significant plaque reduction in the APPSwe mouse model (FIG. 3).
In FIG. 3 the therapeutic efficacy of a high affinity protofibril selective antibody in transgenic mouse model (APPswe) was tested. A: A 14 months old APPSwe transgenic mouse was intracranially injected with PBS and B: high affinity protofibril selective antibody (158) at 1 μg/μl and examined 72 hours following injection. Marked clearance of Aβ burden is noticeable in the subiculum close to the injection site (B; arrow) as compared to the control side (A; arrow).
Example 4
Proximity ligation in combination with high affinity protofibril selective antibody for measurement of Aβ protofibrils. Human wild-type Aβ protofibrils were detected down to 10 pM-range whereas the Aβ monomer preparation were not detected at all. The combination of the hypersensitive proximity ligation method and a high affinity antibody is particularly advantageous since it provides a system to determine only oligomeric forms of the analyte, which is particularly suitable when diagnosing Alzheimer's disease and other protein “aggregation” diseases such as prion disease, Creutzfelt-Jacob, amyloidosis and Parkinson's disease.
In FIG. 4 human Aβ protofibrils are measured at pM levels by the proximity ligation technique. Proximity ligation assay: Method description (from Gullberg et al., 2004): Step 1, incubation of sample with proximity probe pair (≈1 h); step 2, addition of all components required for ligation and detection by quantitative PCR (≈15 min ligation time). A high affinity protofibril selective monoclonal antibody was used in the assay; step 3, quantitative PCR (≈2 h). Synthetic Aβ monomer and Aβ protofibril preparations were diluted and tested for their reactivity in proximity ligation assay described above.
Example 5
mAb 158 does not Recognize a Generic Amyloid Epitope
Previously reported Aβ conformation dependent antibodies have been shown to bind oligomers and fibrils of other amyloidogenic proteins, suggesting a common epitope present on all amyloid aggregates. Due to technical difficulties in generating protofibrils from other amyloidogenic proteins than Aβ, mAb158 was instead tested against different amyloid fibrils. The dot blot assay was used for these experiments since inhibition ELISA, where the antibody-antigen reactions take place in solution, is not suitable for insoluble antigens like fibrils. The dot blot assay is however not suitable for evaluation of antibody specificity for various Aβ forms, i.e. for measuring differences in selectivity for profibrils and fibrils. Fibrils of medin, islet amyloid polypeptide (IAPP) and α-synuclein were immobilized on a nitrocellulose membrane to maintain their native conformations. mAb158 did not exhibit reactivity with any amyloid other the Aβ fibril (FIG. 5A). The binding of mAb 158 to Aβ fibrils suggests that part of the Aβ protofibril epitope is present also in the Aβ fibril structure. As positive controls the antibodies 6E10 (Aβ), pAb179 (medin), pAbA110 (IAPP) and mAb211 (α-synuclein) were used (FIG. 5B). Representative blots from repeated experiments (n=3).
mAb158 does not Bind APP


    
    
        Levels of APP and soluble APP fragments commonly exceed the levels of β in biological samples such as CSF and brain homogenate, and therefore an Aβ-antibody's cross-reactivity to APP could inhibit a treatment by binding to APP, resulting in less free antibody for binding and elimination of Aβ protofibrils and/or Aβ oligomers. Also, it could disturb measurements of Aβ protofibrils in biological samples by a sandwich ELISA assay of Aβ. To elucidate whether mAb158 binds to native APP, immunoprecipitation experiments were performed. HEK-cell culture media (mock, APPSwe and APPArc-Swe) and mouse brain homogenates (non-transgenic, APPWwe and APPArc-Swe) were immunoprecipitated with mAb158 or 6E10, followed by a denaturing Western blot with 6E10 as detecting antibody (FIG. 5C). As seen in FIG. 5C, mAb158 did not immunoprecipitate αAPPs from cell culture media or full length APP from mouse brain homogenates, whereas, as expected, 6E10 did. The synthetic Aβ protofibrils used as control were immunoprecipitated equally well by both antibodies (FIG. 5C). Representative blots from repeated experiments (n=3).
    
    


Example 6
Establishment of an Aβ Protofibril Specific Sandwich ELISA
To enable measurements of Aβ protofibrils in biological samples a sandwich ELISA with mAb158 as both capturing and detecting antibody was established. This assay measures Aβ protofibrils with a detection limit of 1 pM and with a linear range up to 250 pM (FIG. 6A, lines indicate linear regression of the standard curves). Due to uncertainties concerning the size of the Aβ protofibrils used in the standard curve, the concentration 1 pM is based on the molecular weight of one Aβ monomer (4514 g/mol), Though, since the molecular weight of a protofibril has been estimated to be at least 100 kDa, the limit of detection calculated as molar Aβ protofibrils could be as low as 50 fM. A standard curve of AβArc protofibrils gave a lower signal than wild type Aβ protofibrils, possibly due to differences in Aβ protofibril size (FIG. 6A, 6B). Titrated synthetic LMW-Aβ (Low Molecular Weight Aβ). By the term “Low Molecular Weight Aβ”, it is meant monomers, dimers and trimers of Aβ having a molecular weight of approximately 4-12 kDa. Aβ protofibrils and Aβ1-16 were used to validate the conformation specificity of the ELISA (FIG. 6B), where the hydrophilic Aβ1-16 peptide was used since it is not expected to aggregate. An ELISA composed of two identical antibodies requires at least a dimer of a protein to produce a signal and as predicted, Aβ1-16 was not detected with the mAb158 sandwich-ELISA even at μM-concentrations (FIG. 6B). When pre-treating the LMW-Aβ and Aβ protofibrils with 70% formic acid (FA), known to dissociate aggregated Aβ into monomers, the sandwich ELISA the signal was lost (data not shown). Hence, the detection of LMW-Aβ at high nM concentrations (FIG. 6B) is probably due to a small aggregate content of the peptide preparation.
A large excess of monomeric Aβ, holoAPP and APP-fragments, naturally occurring in biological samples, could interfere with the Aβ protofibril analysis by occupying binding sites of the capture antibody coat, thus inhibiting the protofibrils from binding. This problem was investigated by adding an increasing excess of Aβ1-16 to a fixed concentration of Aβ protofibrils (50 pM, expressed as monomer units) and analyzing it with both the mAb158 ELISA and a 6E10-6E10 sandwich ELISA (FIG. 6C). A 500 000-fold molar excess of Aβ1-16, as compared to Aβ protofibrils, did not disturb the measurements with the mAb158 sandwich ELISA, as expected since Aβ1-16 binds poorly to the capture antibody. In contrast, a 500 fold excess of Aβ1-16 was enough to decrease the signal in the 6E10-6E10 ELISA, where Aβ1-16 binds with high affinity to the capture antibody (FIG. 6C). Moreover, when synthetic Aβ protofibrils was added to mock HEK cell culture media or non-transgenic mouse brain homogenates, 90% of the signal was recovered (data not shown).
Example 7
Measurement of Aβ Protofibrils in Biological Samples
The presence of Aβ protofibrils in cell and mouse models carrying the Arctic mutation have been suggested, though until now there has been no method for direct assaying of Aβ protofibrils in biological samples. The mAb158 sandwich ELISA therefore provides the first opportunity to measure Aβ protofibril levels in such cell and mouse models and to compare them to models without this intra-Aβ mutation. Samples from cells and mice carrying only the Swedish mutation were compared to the wild type Aβ protofibril standard curve, whereas samples from cells and mice expressing Aβ with the Arctic mutation were compared to AβArc protofibril standard curve (FIG. 6A). To ensure that all Aβ measured in this assay was in a soluble state, and to exclude any possible interference from Aβ fibrils, all samples were centrifuged for 5 min at 17 900×g before analysis. Groups of cell media from transiently transfected APPWwe and APPArc-Swe HEK-cells were analyzed and compared to mock HEK-cell culture media. Aβ protofibril levels were calculated from the standard curves (FIG. 6A) as the mean value of triplicates and were then normalized to APP levels to compensate for differences in transfection levels (according to Stenh et al.). The Aβ protofibril concentration in APPArc-Swe HEK-cell culture media was 28 pM (±2), significantly higher (p<0.0001) than the 8.2 pM (±0.3) seen in APPSwe (FIG. 7A). No Aβ protofibrils could be detected in mock media. Levels of Aβ protofibrils were also measured in brains from 10 months old APPArc-Swe and APPSwe transgenic mice with both plaques and intraneuronal Aβ pathology (according to Lord et al.). Brains were homogenized in TBS and centrifuged prior to analysis in order to recover the soluble Aβ fraction. Similar to the analysis using cell culture media, Aβ protofibril levels differed significantly (p=0.005) between the groups, with 397 pM (±59) in APPArcSwe and 108 pM (±14) in APPSwe transgenic mouse brains (FIG. 7B).
In the above-mentioned figures (FIGS. 6 and 7) the number of samples were; mock cells (n=3) and transiently transfected with APPWwe (n=8) and APPArc-Swe (n=11). Levels of Aβ protofibrils in APPArc-Swe media were approximately 9 fold higher than in APPSwe media, whereas mock media gave no signal (A). Measurements of Aβ protofibril levels in the TBS-soluble fraction of non-transgenic mouse brain homogenates (n=6) were compared to transgenic mice (APPSwe, n=3, and APPArc-Swe, n=6) (B). Similar to the cell culture media, Aβ protofibril levels of APPArc-Swe mice were 7 fold higher than in APPSwe mice. Error bars show ±SEM.
Example 8
mAb158 Significantly Lowers Aβ Protofibrils and Total Aβ in APPswearc Transgenic Mice after i.p. Administration
mAb158 (12 mg/kg) was injected i.p. once weekly for 18 weeks in 9-10 months old APPswearc mice. After the study, brains were isolated and homogenised in TBS and subsequently centrifuged to sediment insoluble material. The insoluble material was solubilised in formic acid. Hence, two fractions were obtained from mouse brains i.e. a TBS fraction and a formic acid fraction. Aβ protofibril levels in the TBS fractions were determined by an ELISA. A significant reduction of Aβ protofibrils was found in the mAb158 treatment group compared to the placebo group (FIG. 8). FIG. 8 shows the Aβ protofibril levels in APPswearc transgenic mouse brain TBS extracts after 4 months treatment with either mAb158 or placebo.
Total Aβ in the formic acid fraction was determined by an ELISA (the formic acid was used to solubilise all Aβ forms, in order to make all Aβ forms detectable). A significant reduction of total Aβ was observed in the treatment group compared to the placebo group (FIG. 9). FIG. 9 shows the total Aβ levels in APPswearc transgenic mouse brain formic acid extracts after 4 months treatment with either mAb158 or placebo.
Examples 9-11
Abbreviations


    A Adenine
    Ab protocol AERES biomedical protocol
    BHK baby hamster kidney
    bp base pairs
    C Centrigrade
    C Cytosine
    CHO Chinese Hamster Ovary
    CMF Calcium and Magnesium Free
    COS 7 African green monkey kidney fibroblast cell line
    dhfr Dihydrofolate-reductase
    DMEM Dulbecco's Modified Eagles Medium
    DMSO Dimethyl sulphoxide
    DNA Deoxyribonucleic acid
    ELISA Enzyme linked immuno-adsorbent assay
    FCS Foetal Calf Serum
    g grams
    G Guanine
    hr hour
    HRP Horseradish peroxidase
    IgG Immunoglobulin
    K G or T (IUPAC convention)
    LSAP Large Soluble Amyloid Product
    mAb monoclonal antibody
    sec second
    min minute
    M A or C (IUPAC convention)
    MTX Methotrexate
    NIMR National Institute for Medical Research (UK)
    nm nanometer
    OD optical density
    PBS Phosphate Buffered Saline
    PCR Polymerase chain reaction
    R A or G (IUPAC convention)
    RT Room Temperature
    S C or G (IUPAC convention)
    T Thymine
    UV Ultra Violet
    V variable
    V A or C or G (IUPAC convention)
    VH Immunoglobulin heavy chain variable region
    VK Immunoglobulin kappa light chain variable region
    W A or T (IUPAC convention)
    Y C or T (IUPAC convention)


Materials












Equipment
UK Supplier
Catalog Number







DNA thermal cycler: GeneAmp 9600
Perkin Elmer
N801-0177


A designated tissue culture laboratory
Walker Safety Cabinets Ltd.
N/a


containing a class II microbiological safety




cabinet fitted with a UV-lamp




Innova ® bench top incubator shaker
New Brunswick Scientific
4000


Bench top centrifuge
Fisher Scientific
CEK-126-010N


CO2-gassed 37° incubator
RossLab plc
HSO-501TVBB


Microbiological incubator
Kendro/Heraeus
B6060


Electroporator Model: Gene Pulser II
Bio-Rad Laboratories Ltd.
341BR-3092


ELISA reader: Microplater Reader 3550
Bio-Rad Laboratories Ltd.
3550


Microplate Manager ® 2.2 data analysis
Bio-Rad Laboratories Ltd.
N/a


software package for Macintosh computer




96-Well GeneAmp PCR System 9700
ABI
N8050200


ABI PRISM 310 Genetic Analyzer
Applied Biosystems
310-00-100/120


T100 surface plasmon resonance detector
Biacore









Plastic Consumables













Article
UK Supplier
Catalog Number







175 cm2 tissue culture flask
Sarstedt Ltd
83.1812.002


25 cm2 tissue culture flask
Corning Costar
 3056


30 ml universal container
Sterilin
128C


75 cm2 tissue culture flask
Sarstedt Ltd
83.1813.002


Electroporation cuvettes
Bio-Rad Laboratories Ltd.
165-2088


ELISA plates: Nunc MaxiSorp
Invitrogen Life Technologies
43945A


GeneAmp TM PCR reaction tubes
Perkin Elmer
N801-0180


Glasstic ® disposable cell-counting slide
Bio-stat Diagnostic
887144


Nunc inoculating needles
Life Technologies
254399


tissue culture petri 100 × 20 mm, multi-vent
Helena Biosciences
 93100


tissue culture plate: 6-well + lid
Corning
C3516


tissue culture plate: 24-well + lid
Corning
C3526









Immunology and Molecular Biology Reagents














Article
UK Supplier
Catalog No.
Lot No.


















1st strand synthesis kit
Amersham Biosciences
27-9261-01
3375313


Advantage ®-HF 2 PCR Kit
Clontech
639123
6040151


Agarose (UltraPure TM)
Invitrogen
15510-027
3019491


Albumin bovine (BSA)
Calbiochem
126575
B65755


Ampicillin
Sigma
A-9518
63H0992


Apa I
Promega
R636
16007003


Themoprime+ DNA Polymerase
Abgene
AB0301
014/0103/11





019/0607/13





020/1808/13


Bam HI
Promega
R602
15851606


BigDye ® Terminator v3.0 Cycle
ABI
4390242
0605143


Sequencing Ready Reaction Kit


0608154


Ethidium Bromide (10 mg/ml)
Sigma
E-1510
43H9414


Goat anti-human IgG (Fc fragment
Stratech Scientific
109-005-098
68215


specific) antibody





Goat anti-human kappa chain
Sigma
A7164
032K9157


horseradish peroxidase conjugate





Hind III
Promega
R604
16834803


Human IgG1/kappa antibody.
The Binding Site
BP078
223729


K-Blue HRP substrate
SkyBio
308176
060823


Oligonucleotides
Sigma
n.a.



PBS Tablets
Sigma
P4417
11K8204


QIAGEN Plasmid Maxi Kit (25)
Qiagen
12162
124114870


QIAprep Spin Miniprep Kit
Qiagen
27106
124117906


QIAquick gel purification kit
Qiagen
28704
11549740


QIAquick PCR purification kit
Qiagen
28106
G10.1.12


Red Stop Solution (For K Blue)
SkyBio Ltd,
301475
060104



Qiagen
74106
10916587


Shrimp alkaline phosphatase
USB
70092Y
107635


Subcloning Efficiency TM DH5α TM
Invitrogen
44 0098
1164658


Chemically Competent E. coli





T4 DNA Ligase
Promega
M1801
167080


TMB One-Step substrate for HRP
SkyBio Ltd,
KB176



TOPO-TA Cloning ® kit
Invitrogen
45-0641
1350772


X-Gal
Sigma
B-9146
20965701









Solutions from National Institute of Medical Research
















Solution name:
Components
Amount





















PBS ‘A’ Dulbeccos (Ca &
NaCl
8 
g



Mg Free)

0.2
g




KCl
1.15
g





0.2
g




Na2HPO4
1
L




KH2PO4






water





LB
Bacto Tryptone
10
g




Yeast Extract
5
g




NaCl
10
g




water
1
L



LB agar
LB
1
L




Agar (Difco)
15
g










Culture Reagents

















Catalog




Article
UK Supplier
Number
Lot Numbers
Expiry date







DMEM (1X) Dulbecco's Modified
Invitrogen
41966-047
  9206
July 2007


Eagle Medium (High glucose) with






GlutaMAX TM I, 4500 mg/L D-






Glucose, Sodium Puruvate






DMSO (Dimethyl sulfoxide)
Sigma
D2650
125K2409
December 2007 


Penicillin & Streptomycin
lnvitrogen
15070-063
1298401



Serum: Fetal Clone I
Perbio
SH30080
AMM17779
December 2007



Science





SOC
Invitrogen
15544-034
1306051



Trypan Blue
Sigma
T8154
19H2388



Trypsin-EDTA solution, cell culture
Sigma
T4049
48K2342
April 2008


tested, 0.25%









Example 9
DNA Sequence of 158 Antibody
9.1—RNA Preparation
Snap-frozen cell pellets of the mouse hybridoma 158, (labelled vials 060824#158 5×106 cells) were received by TAG on Oct. 3, 2006. These cells were stored frozen until processing using the Qiagen RNeasy midi kit to isolate RNA following the manufacturers protocol.
9.2 —1′′ Strand Synthesis
About 5 micrograms of 158 RNA was subjected to reverse transcription to produce 158 cDNA using the Amersham Biosciences 1st strand synthesis kit following the manufacturers protocol—This was repeated to generate 3 independent cDNA products (rounds 1, 2 and 3) in order to obviate DNA mutations due to the RT reaction.
9.3 Cloning of the 158 Immunoglobulin cDNA


    
    
        Hybridoma 158 cDNA was amplified by PCR in 23 separate reactions. Immunoglobulin kappa chain variable region (VK) cDNA was amplified using 11 VK primers (MKV1-11) in combination with the kappa constant region primer MKC (Table 6). Similarly, immunoglobulin heavy chain variable region (VH) cDNA was amplified by PCR using 12 different VH primers (MHV1-12) in combination with a mix of the four IgG constant region primers (MHCG1/2a/2b/3: Table 7).
    
    


The result of the initial set of IgH PCR reactions was the single amplification product using MHV5 primer. None of the other 11 primer pairs gave a PCR product. The product of the PCR reaction primed by the oligonucleotide primers: MHV5+(MHCG1/2a/2b/3 mixture) was ligated into the pCR2.1®TOPO® vector using the TOPO-TA Cloning® kit. The result of the initial set of IgK PCR reactions was two single amplification products using primers MKV1 and MKV2 with MKC. The other 9 primer pairs generated no product. The products of the PCR reaction primed by the oligonucleotide primers: MKV1 or MKV2+MKC were ligated into the pCR2.1®-TOPO® vector using the TOPO-TA Cloning® kit. E. coli TOP10 bacteria transformed with the ligated vector were cloned on LB/ampicillin/X-gal agar plates, by picking onto agar grid and into PCR screening mixture. The cloned plasmid inserts were screened by PCR amplification. The PCR products were gel electrophoresed and clones producing the correct-sized PCR amplification product (500 bp approx) were identified. Overnight cultures (5 ml) of each clone were processed using the QIAprep Spin Miniprep Kit Protocol, to produce DNA plasmid minipreps.
9.4—cDNA Sequence Determination
The complete cycle of RT-PCR, cloning, and DNA sequence analysis was repeated to obtain three completely independent sets of sequence information for each immunoglobulin chain. Plasmid clones from each independent set of RT-PCR reactions were sequenced in both directions using the 1212 and 1233 primers (Table 10). Plasmids were sequenced using the BigDye® Terminator v3.0 Cycle Sequencing Ready Reaction Kit (ABI), cycled on a GeneAmp9600 PCR machine and analysed on an ABI 310 capillary sequencer.
9.5-158 VK DNA Sequence
Sequences of VK clones generated using PCR primers MKV2 and MKC on 1st strand cDNAs rounds 1 and 2, were identical to a sterile kappa transcript originating from the myeloma fusion partner such as MOPC-21, SP2 and Ag8. This is a sterile transcript.
The consensus sequence (158 VK) of VK clones generated using PCR primers MKV1 and MKC on 1st strand cDNAs rounds 1-3 is shown in Table 11. This is a functional rearrangement. Table 11 shows some differences from the sequence shown in Tables 1, 4 and 5. These differences are in the FW1 region where the PCR primer was located. The mouse VK leader sequence most identical to the fragment of leader in 158 VK, not encoded by our primers, was K5.1# (Table 12). The prediction for the signal peptide to cleave correctly the #K5.1 signal sequence was done by a prediction program. Most likely predicted cleavage site was correctly between amino acid residue 19 and 20. (Table 13; FIG. 15). The chimeric 158VK protein and DNA sequence is shown in Table 14.
9.6-158 VII DNA Sequence
The consensus sequence (158 VH) of VH clones generated using PCR primers MHV5 and MHCG1/2a/2b/3 mixture on 1st strand cDNAs rounds 1-3 is shown in Table 15. As with 158 VK, there are some differences from the FW1 sequence shown in Tables 1, 4 and 5. The most identical mouse VH leader sequence to the fragment of leader, not encoded by our primers, was NL-1 (Table 16).
Example 10
Construction of Chimeric Expression Vectors
Construction of chimeric expression vectors entails adding a suitable leader sequence to VH and VK, preceded by a Hin dIII restriction site and a Kozak sequence. The Kozak sequence (Table 8) ensures efficient translation of the variable region sequence. It defines the correct AUG codon from which a ribosome can commence translation, and the most critical base is the adenine at position −3, upstream of the AUG start. The leader sequence is selected as the most similar mouse leader sequence in the Kabat database. These additions are encoded within the forward primers (Table 9). Furthermore, the construction of the chimeric expression vectors entails introducing a 5′ fragment of the human γ1 constant region, up to a natural Apa I restriction site, contiguous with the 3′ end of the J region of 158. The CH is encoded in the expression vector downstream of the inserted VH sequence but lacks the V-C intron. For the light chain, the natural splice donor site (Table 8) and a Bam HI site is added downstream of the V region. The splice donor sequence facilitates splicing out the kappa V:C intron which is necessary for in-frame attachment of the VK to the constant region.
The mouse VH and VK genes were analysed to identify any unwanted splice donor sites, splice acceptor sites, Kozak sequences and for the presence of any extra sub-cloning restriction sites which would later interfere with the subcloning and/or expression of functional whole antibody. In this case none were found.
10.1—Expression Vectors
Plasmid DNA preparations of the expression vectors pKN100, and pG1D200 were purified using Qiagen Maxi kits following the manufacturers protocol. Plasmid DNA Purification using QIAGEN Plasmid Midi and Maxi Kits, from 500 ml cultures of TOP10 bacteria transfected with either vector. The vector maps are shown in FIGS. 10 and 11.
10.2—The Light Chain Chimerisation Primers
The mouse leader sequence K5.1# was incorporated into the design of the chimeric 158 VK. Primers were designed to generate a PCR product containing this complete leader, and 158 VK, with terminal restriction sites Hind III and Bam HI for cloning into the pKN100 expression vector (Table 9). The forward primer 158v1 introduces a Hind III restriction site; a Kozak site and the K5.1# leader sequence. The back primer 158v1rev introduces: a splice donor site and a Bam HI restriction site.
10.3—The Heavy Chain Chimerisation Primers
The leader sequence NL-1 was incorporated into the design of the chimeric 158 VII. Primers were designed to generate a PCR product containing this leader, and the 158 VH region, with terminal restriction sites Hin dIII and Apa I for cloning into the pG1D200 expression vector.
These are shown in Table 9. The forward primer, 158vh, introduces a Hin dIII restriction site; a Kozak translation initiation site and the NL-1 leader sequence. The back primer, 158vhrev, introduces the 5′ end of the γ1 C region and a natural Apa I restriction site. The signal peptide cleavage site prediction for K5.1 leader sequence of VK is shown in Table 17 and FIG. 16.
10.4—Generation of the Chimeric 158 VH Construct: pG1D200158VH
The 158 VH DNA fragment was amplified with primers: 158vh and 158vhrev (Table 9). The 450 bp (approx) PCR product was T-A ligated into the vector pCR2.1 and used to transform chemically competent TOP10 bacteria. Clones were selected by appropriate insert size and sequenced using the 1212 primer (Table 10). The correct expression insert was subcloned into pG1D200 expression vector and the correct subclone was selected by DNA sequencing using primer BDSH61R (Table 10). This clone was grown in 200 ml culture to produce plasmid DNA using the Qiagen Maxi Kit using the manufacturers protocol. The chimeric 158VH protein and DNA sequence is shown in Table 18.
10.5—Generation of the Chimeric 158 VK Construct: pKN100158VK
The 158 VK DNA fragment was amplified with primers 158v1 and 158v1rev (Table 9). The 450 bp (approx) PCR product was T-A ligated into vector pCR2.1 and used to transform chemically competent TOP10 bacteria. Clones were selected by insert size and sequenced using the 1212 primer (Table 10). The correct clone was subcloned into pKN100 expression vector. The correct subclone was selected by screening for insert size and DNA sequencing using primer Hu-K2 (Table 10). This clone was grown in 200 ml culture to produce plasmid DNA using the Qiagen Maxi Kit using the manufacturers protocol.
Example 11
Production and Binding Properties of Chimeric 158 Antibody
11.1—COS 7 Cell Transformation and Cell Culture
One vial of COS 7 cells was thawed and grown in DMEM supplemented with 10% Fetal clone 1 serum and antibiotics. One week later, cells (0.8 ml at 107/ml) were electroporated with pG1D200158VH plus pKN100158VK (10 μg DNA each). The cells were grown in 8 ml of growth medium in petri dishes for 3 days.
11.2—Chimeric Antibody Production
A sandwich ELISA was used to measure antibody concentrations in the COS 7 supernatants. Chimeric 158 VH×158 VK antibody was expressed at 0.3 μg/ml and subsequently at 3.7 μg/ml (Table 19) in transiently co-transfected COS cell conditioned media.
11.3—Chimeric Antibody Activity
Two ELISAs was used to analyse the antigen binding of chimeric 158. Using the 3.7 μg/ml chimeric antibody conditioned medium, binding to Aβ monomer was measured by a direct ELISA protocol (FIG. 12) and compared to the mouse 158 IgG. Secondly, a competition ELISA was done using either monomer or protofibril mixed in the fluid phase with antibody, which subsequently bound to Aβ monomer in the solid phase (FIG. 13). These showed that the chimeric 158 antibody binds to amyloid Aβ monomer and protofibril similarly to the original 158 mouse antibody.
Comment
Later sequencing has shown that the mouse antibody sequence data, as shown in Tables 1 and 4 contain errors in both VH and VK chains at the 5′ end. We suggest that this is due to the use of primers located within the V region. In later sequencing, primers located within the leader sequences, which cannot introduce mutations within the V regions, were used. The later sequencing showed sequence differences (see Tables 15 and 11). Said differences are however not located within the CDR regions.
The chimeric antibody binds amyloid Aβ monomer and protofibrils as shown by the direct binding ELISA and the competition ELISA respectively. This evidence confirms that the combination of 158 VH and 158 VK chains encodes the anti-LSAP antibody 158 and indicates that these sequences are suitable for the humanisation procedure to generate a humanised 158 antibody.
Example 12
Humanised Antibody Design and Discussion
Abbreviations and Definitions


    158 mouse monoclonal anti-LSAPTM antibody 158
    158 VH VH of mouse 158 antibody
    158 VK VK of mouse 158 antibody
    158RKAss Humanised version of 158 VK retaining cryptic splice sites
    158RKA Humanised version of 158 VK with cryptic splice sites removed
    158RHAss Humanised version of 158 VH retaining cryptic splice sites
    158RHA Humanised version of 158 VH with cryptic splice sites removed
    A Adenine
    bp base pairs
    C Cytosine
    CDR Complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering system
    D-gene Diversity gene
    DNA Deoxyribonucleic acid
    FW Framework region: the immunoglobulin variable regions excluding the CDR regions
    G Guanine
    IgG immunoglobulin G
    J-gene Joining gene
    Kabat an immunoglobulin alignment and numbering system pioneered by Elvin A Kabat
    mAb monoclonal antibody
    MRCT Medical-Research Council Technology
    T Thymine
    VCI Framework residue classified as vernier or canonical or VH-VL interface
    V-gene The gene segment that is rearranged together with a J (and D for VH) gene to generate a complete VH or VK
    V region The segment of IgG chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.
    VH Immunoglobulin heavy chain variable region
    VK Immunoglobulin kappa light chain variable region

Equipment














Hardware & software
Origin







SGW02 computer
Silicon Graphics


PC computer
Hewlett Packard


SR 7.6
Steve Searle, Wellcome Trust Sanger Institute,



Cambridge.


Lasergene 6.0
DNAstar Inc


Modeler 9.0
Accelrys Ltd.


SignalP
Center for Biol. Sequence Analysis, Technical



University of Denmark Website


BlastP
NCBI website









12.1—Human V Gene Databases
The protein sequences of human and mouse immunoglobulins from the International Immunogenetics Database 2006 and the Kabat Database Release 5 of Sequences of Proteins of Immunological Interest (last update 17 Nov. 1999) were used to compile a database of immunoglobulin protein sequences in Kabat alignment. Our database contains 9322 human VH and 2689 human VK sequences. The sequence analysis program, SR 7.6, was used to query the human VH and VK databases with 158 VH and 158 VK protein sequences (Table 20).
12.2—Selection of a Human Framework for 158RHA
12.2.1—Comparison of 158 VH with Human VH Sequences
Human VH sequences with highest identity to 158 VH at Vernier (Foote, J. and G. Winter. 1992. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol. Biol. 224:487-499.), Canonical (Morea, V., A. M. Lesk, and A. Tramontano. 2000. Antibody modeling: implications for engineering and design. Methods 20:267-279.) and VH-VL Interface (Chothia, C., J. Novotny, R. Bruccoleri, and M. Karplus. 1985. Domain association in immunoglobulin molecules. The packing of variable domains. J Mol. Biol. 186:651-663.) (VCI) residues, located within the V-region framework (FW), are shown in Table 21. The number of VCI residues (VCI score) and FW residues (FW score) identical to 158 are also shown. All these VH sequences share identical VCI residues, and CDR lengths, as shown in Table 22. AJ556669 has an unusual Pro74 not seen in the other human sequences in this dataset, leading us to discount it in the initial analysis. Pro74 is, however, present in the 158VH sequence, so AJ556669 could be considered as an alternative FW for humanisation, if the VH construct based on AF062243 does not bind antigen. The alignment of these sequences (Table 23) highlights their differences. AF062243 uniquely within this dataset has the conservative change T(82a)S and the conservation of F79. The other features of AF062243 are the conservative changes D1E, K19R, A23S, T77S, S118T. All other FW changes were common to all the frameworks in Table 23. AF062243 was selected as the framework on which to base 158RHA.
12.3—Generation of 158RHA
The design of 158RHA is simply the grafting of CDR 1, 2 and 3 from 158 VH into the acceptor FW of AF062243. The human germline V-gene most identical to AF062243 is VH M99649 (VH3-07), (Table 24) from which the leader peptide was extracted (Table 25). The SignalP algorithm (Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1-6.) predicted that it would cut appropriately with signal peptidase (Table 26; FIG. 17). Table 27 shows the scheme of grafting 158 VH CDR 1, 2 and 3 into the AF062243 FW, to generate 158RHA protein sequence. Table 28 shows the generation of the DNA sequence 158RHAss from the natural DNA sequences of 158 VH and AF062243. Analysis of the 158RHAss DNA sequence predicted the presence of splice donor sites, the prediction scores of which are shown in Table 29. Non-coding mutations were introduced to inactivate these predicted splice sites, as shown in Table 30 to generate the final 158RHA DNA sequence (Table 31).
12.4—Selection of a Human Framework for 158RKA
12.4.1—Comparison of 158 VK with Human VK Sequences
The human VK sequences with highest identity to 158 VK at VCI residues are shown in Table 32 together with the number of VCI residues (VCI score) and FW residues (FW score) identical to 158 VK. Eleven sequences have all VCI residues identical to 158 VK. Table 33 shows that all these sequences have CDR lengths identical to 158 VK. Table 34 highlights their differences, showing that K45 is retained in AB064054 only, which also retains 185. The G100P change is unremarkable because P100 is common, having an incidence of 15% in our human VK database. The two substitutions: T7S and K74R, are conservative, and all other substitutions are common to all the sequences in Table 34. For these reasons AB064054 was selected to generate 158RKA.
12.5—Generation of 158RKA
The design of 158RKA is the simple grafting of the CDRs 1, 2 and 3 from 158 VK into the acceptor FW of human AB064054. The nearest germline V-gene to AB064054 is A19 (Table 35), from which the leader peptide was extracted (Table 36). The SignalP algorithm predicted appropriate cutting (Table 37; FIG. 18) of this leader peptide. Table 38 shows the generation of the protein sequence of 158RKA by intercalation of the 158 VK CDRs into the FW of AB064054. Table 39 shows the generation of the DNA sequence of 158RKAss from the natural DNA sequence of 158 VK and AB064054. Analysis of the 158RKAss predicted the presence of splice donor sites, the scores of which are shown in Table 40. Non-coding mutations (41) were introduced to inactivate these sites and generate the final 158RKA DNA construct (Table 42).
12.6 Humanized Antibody (BAN2401) Binding Activity
The 158RKA and 158RHA genes were inserted into an expression vector containing the IgG1 constant region. This construct was expressed in COS cells to generate the humanized 158 antibody. The humanized 158 antibody was tested for binding activity and specificity in a competitive ELISA. The humanised antibody exhibited identical binding properties as to mAb158 and the 158 chimeric antibody (see FIG. 14.)
12.7 Additional Mutations in the 158RHA and 158RKA Chains
By comparing mouse germline V genes VH AAK71612 to 158 VH a single somatic mutation A60G in the CDR2 was identified. Furthermore, the molecular model of antibody 158 which contains three VH FW residues within 5 Å of CDR residues which are unconserved in 158RHA. These substitutions are DIE, P74A and T82S (Table 43). Similarly, there are two VK FW residues within 5 Å of CDR residues which is unconserved in 158RKA. This substitution is L3V and G100P (Table 44). Introduction of back mutations at positions VH-1, VH-74, VH-82, VK-3 and VK-100 into 158RHA and 158RKA, in humanised versions 158RHB, 158RHC, 158RHD, 158RKB and 158RKC are shown in Table 43 and 44.
REFERENCES


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    Bard et al., Nat. Med. 6:916-919, 2000.
    Bayer et al., Neurology 64:94-101, 2005.
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    Hoshi et al. Proc. Natl. Acad. Sci, 100:6370-6375, 2003.
    Jarret J. T., Biochemistry, 32, 4693-4697,1993.
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    Pfeifer et al., Science 298:1379, 2002.
    Racke et al., J. Neurosci 25:629-36, 2005.
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TABLE 1





Amino acid sequence of variable regions of the heavy chain (VH) and light chain (VL/Vκ)


from six different monoclonal antibodies specific for human wild-type Aβ protofibrils.
















VH-EA1: X731
EVKLVESGGGLVGPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYVADTVKGRFTISRDNPKN



TLPLQWLSLRESRDTAMYYCARYGWYAM-----DYWGQGTSVTVSS



SEQ ID NO: 13


 


VH-EA2: X736
EVELVESGGGLVKPSGSLKLSCAASGFTPSSYAMSWVRQTPEKRLEWVATISSGGSYTYYPDSVRGRFTISRDKAKN



TLYQKSSLRSRDTAMYYCARNYGSRRYF-----DVWGAGTSVTVSS



SEQ ID NO: 14


 


VH-EA3: X745
QVELQQSGPELVKPSASVKMSCKASGVTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKPKGKATLTSDKSSSV



AYNCLSSLRSEDSAVYYCARRVSPLTSYAM---DYWGQGTSVTVSS



SEQ ID NO: 15


 


VH-EA7: X746
QVQLKESGPSLNAPPQSLSITCTVSGFSLTSYGVHWNRQPPGKGLEWLGVIWAGGSTNYNSALMS-RLSISKDNSKSQ



VFLKNNSLQTDDTAMYYCARGRYDGKTRFA-----YWGQGTLVTVSS



SEQ ID NO: 16


 


VH-EA8: X748
EVKLMESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGR



FTISRDNPKNTLFLWMTSLREEDTAMYYCARGDSF---------DYWGQGTTLTVSS



SEQ ID NO: 17


 


VH-EA9: X758
EVQRVESGGGLVGPSGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNPKNTL



ELQWTSLRSEDTAMYYCAREGGYYYGRSYYTMDYWGQGTSVTVSS



SEQ ID NO: 18


Vκ-BA1: X731
DVVMTQTPLSLPVSLGDCASLSCRSSQSIVHSNGMTYLE-WYLQKPGQSPKLLINKVSNRFSGVPDRFSGSGSGTDFTL



KISRVEAEDLGVYYCPQGSHVPPIGGGGTKLRIK



SEQ ID NO: 19


 


Vκ-BA2: X736
DIVMTQAPKFLLVSAGDRVTITCKASGSVSNDVA------WYQQEPGDSPKLLIYYASNAYTGVPDRFTGSSY



GTDFTFTISTVQAEDLAVYFCQQDYSSPFTFGSGTKLEIK



SEQ ID NO: 20


 


Vκ-BA3: X745
DIVMTQAFSSLAVSAGEKVTMSCKSSQSLLNSRTRENYLAWYQQKPGQSPKLLIYWASTRESGVFDRFTGSGS



GTDFTLTISSVQAEDLAVYYCKQSYNL--WTFGGGTKLEIK



SEQ ID NO: 21


 


Vκ-BA7: X746
EWVLTQSPAEMSASPGEKVTMTCRASESVSSEYLH-----WYQQKSGASPKLWIYSTSNLASGYPARFSGSSS



GTSYGLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELK



SEQ ID NO: 22


 


Vκ-BA8: X748
DIVMTQAFLSLFVSLGDQASISCRSSQSLVHSNGNTYLH-WYLQKPGQSLKLLIYKVSNRFSGVPDRFSGGGSG



TDFTLKISRVEAEDLGYYECSQSTHVPLTFGAGTKLELK



SEQ ID NO: 23


 


Vκ-BA9: X758
DIVWTQAPLSLPVSLGDQASISCRSSQSIVHWNGQTYL-EWYLQEPGQSPKLLIVKVSNRFSG



VPDRFSGSGSGTDFTLKISRVEADLGIYYCFQGSHVPPTFGGGTKLEIK



SEQ ID NO: 24





*Position of the various CDR regions (1-3) are underlined in VL and VH. The boundaries of the CDR regions


(1-3) are shown in Table 3 and Table 4. Antibody BA9, also named 158 in the patent application., is an


example of a high affinity protofibril specific antibody according to the invention.













TABLE 2





Amino acid sequences of CDR1-2 regions from VH chain from a


protofibril selective antibody and amino acid substitutions


that are compatible with high affinity binding to human


wild-type AB protofibrils.

















VH chain CDR-1 region




AASGFTFSSFGMHWVR
(SEQ ID NO: 25)
Antibody 158


 


---------YA-S---
(SEQ ID NO: 26)
Substitutions*


 


VH chain CDR-2 region




WVAYISSGSSTIYYGDTVKGRFT
(SEQ ID NO: 27)
Antibody 158


 


--------------A--------
(SEQ ID NO: 28)
Substitutions*


 


---T----G-YT--P-S------
(SEQ ID NO: 29)
Substitutions*


 


VH chain CDR-3 region




CAREG-GYYYGRSYY-TMDYWGQ
(SEQ ID NO: 30)
Antibody 158


 


CARYGxxxxxNYxxxxAMDYWGQ
(SEQ ID NO: 31)
Substitutions and deletions*


 


CARNYxxxxGSRRxxxYFDVWGA
(SEQ ID NO: 32)
Substitutions and deletions*





*The amino acid substitutions (other amino acid than in antibody 158) are shown with one amino acid letter code. Deletions are shown with (x).













TABLE 3





Amino acid sequences of CDR 1-3 regions from VL chain from a


protofibril selective antibody and amino acid substitutions


that are compatible with high affinity binding to human


wild-type AB protofibrils

















VL chain CDR-1 region




ISCRSSQSIVHSNGNTYLEWYL
(SEQ ID NO: 33)
Antibody 158


 


ITCKASQSVxxSNDxxxVAWYQ
(SEQ ID NO: 34)
Substitutions and deletions*


 


VL chain CDR-2 region




LIYKVSNRFSGVP
(SEQ ID NO: 35)
Antibody 158


 


---YA---YT---
(SEQ ID NO: 36)
Substitutions*


 


VL chain CDR-3 region




YYCFQGSHVPPTFGG
(SEQ ID NO: 37)
Antibody 158


 


-F-Q-DYSS-F---S
(SEQ ID NO: 38)
Substitutions*





*The amino acid substitutions (other amino acid than in antibody 158) are shown with one amino acid letter code. Deletions are shown with (x).













TABLE 4





Amino acid sequence of mouse framework regions of the mouse and human variable


light chain (VL) region from protofibril specific antibodies
















Mouse framework* VL regions



Divmtqaplslpvslgdqasiscwylqkpgqspklliygvpdrfsgsgsgtdftlkisrveaedlgiyyc
antibody 158


(SEQ ID NO: 39)



......................................................................
BA9_VL_fr123


(SEQ ID NO: 40)



.v....t...........................................................v...
BA1_VL_fr123


(SEQ ID NO: 41)



........kf.l..a..rvt.t...q..................t...y.....ft..t.q....av.f.
BA2_VL_fr123


(SEQ ID NO: 42)



 


Human framework VL regions



......t......tp.ep...............q..............................v.v...
VKII-3-1-(1)-O11


(SEQ ID NO: 43)



......s......tp.ep...............q..............................v.v...
VKII-4-1-(1)-A19


(SEQ ID NO: 44)



......t....s.tp.qp...............q..............................v.v...
VKII-4-1-(1)-A18


(SEQ ID NO: 45)



......t....s.tp.qp.............p.q..............................v.v...
VKII-4-1-(1)-A2


(SEQ ID NO: 46)



.v....s......t..qp......fg.r.....rr.............................v.v...
VKII-4-1-(1)-A17


(SEQ ID NO: 47)





*Framework region is the region outside the CDR regions. The CDR regions has been deleted for clarity.













TABLE 5





Amino acid sequence of mouse and human framework regions of the mouse and human


variable light heavy (VH) region from protofibril specific antibodies
















Mouse framework* VH regions



Evklmesggglvqpggsrklscaaswvrqapekglewvarftisrdnpkntlflqmtslrsedtamyycar
antibody 158


(SEQ ID NO: 48)



.......................................................................
BA9_VH_fr123


(SEQ ID NO: 49)



....v..................................................................
BA1_VH_fr123


(SEQ ID NO: 50)



....v.......k....l...........t...r.............a....y...s..............
BA2_VH_fr123


(SEQ ID NO: 51)



 


Human framework VH regions



..q.v............lr............g...............a..s.y...n...a....v.....
VH3-7_fr123


(SEQ ID NO: 52)



..q.v............lr............g......s........s....y...n...a....v.....
VH3-53_fr123


(SEQ ID NO: 53)



..q.v............lr............g......s........s....y...n...a....v....k



(SEQ ID NO: 54)



..q.v............lr............g......s........a..s.y...n...d....v.....
VH3-48_fr123


(SEQ ID NO: 55)



..q.v............lr............g...v..s........a....y...n...a....v.....
VH3-74_fr123


(SEQ ID NO: 56)





*Framework region is the region outside the CDR regions. The CDR regions has been deleted for clarity.













TABLE 6







PCR primers for cloning mouse VK










Name
Sequence (5′→3′)







MKV1
ATGAAGTTGVVTGTTAGGCTGTTGGTGCTG




(SEQ ID NO: 57)



 



MKV2
ATGGAGWCAGACACACTCCTGYTATGGGTG




(SEQ ID NO: 58)



 



MKV3
ATGAGTGTGCTCACTCAGGTCCTGGSGTTG




(SEQ ID NO: 59



 



MKV4
ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG




(SEQ ID NO: 60)



 



MKV5
ATGGATTTWAGGTGCAGATTWTCAGCTTC




(SEQ ID NO: 61)



 



MKV6
ATGAGGTKCKKTGKTSAGSTSCTGRGG




(SEQ ID NO: 62)



 



MKV7
ATGGGCWTCAAGATGGAGTCACAKWYYCWGG




(SEQ ID NO: 63)



 



MKV8
ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG




(SEQ ID NO: 64)



 



MKV9
ATGGTRTCCWCASCTCAGTTCCTTG




(SEQ ID NO: 65)



 



MKV10
ATGTATATATGTTTGTTGTCTATTTCT




(SEQ ID NO: 66)



 



MKV11
ATGGAAGCCCCAGCTCAGCTTCTCTTCC




(SEQ ID NO: 67)



 



MKC
ACTGGATGGTGGGAAGATGG




(SEQ ID NO: 68)

















TABLE 7







PCR primers for cloning mouse heavy VH










Name
Sequence (5′→3′)







MHV1
ATGAAATGCAGCTGGGGCATSTTCTTC




(SEQ ID NO: 69)



 



MHV2
ATGGGATGGAGCTRTATCATSYTCTT




(SEQ ID NO: 70)



 



MHV3
ATGAAGWTGTGGTTAAACTGGGTTTTT




(SEQ ID NO: 71)



 



MHV4
ATGRACTTTGGGYTCAGCTTGRTTT




(SEQ ID NO: 72)



 



MHV5
ATGGACTCCAGGCTCAATTTAGTTTTCCTT




(SEQ ID NO: 73)



 



MHV6
ATGGCTGTCYTRGSGCTRCTCTTCTGC




(SEQ ID NO: 74)



 



MHV7
ATGGRATGGAGCKGGRTCTTTMTCTT




(SEQ ID NO: 75)



 



MHV8
ATGAGAGTGCTGATTCTTTTGTG




(SEQ ID NO: 76)



 



MHV9
ATGGMTTGGGTGTGGAMCTTGCTATTCCTG




(SEQ ID NO: 77)



 



MHV10
ATGGGCAGACTTACATTCTCATTCCTG




(SEQ ID NO: 78)



 



MHV11
ATGGATTTTGGGCTGATTTTTTTTATTG




(SEQ ID NO: 79)



 



MHV12
ATGATGGTGTTAAGTCTTCTGTACCTG




(SEQ ID NO: 80)



 



MHCG1
CAGTGGATAGACAGATGGGGG




(SEQ ID NO: 81)



 



MHCG2a
CAGTGGATAGACCGATGGGGC




(SEQ ID NO: 82)



 



MHCG2b
CAGTGGATAGACTGATGGGGG




(SEQ ID NO: 83)



 



MHCG3
CAAGGGATAGACAGATGGGGC




(SEQ ID NO: 84)







Legend: Wobble bases are defined in Abbreviations (Section 2).













TABLE 8







Sequences important for efficient


expression of immunoglobulin in mammalian cells








Name
Consensus DNA Sequence (5′→3′)





Kozak trans-
G C C G C C R C C−1 A+1 U G G


lation initia-
(SEQ ID NO: 85)


tion site



 


Kappa light
A C :: G T R A G T


chain splice
(SEQ ID NO: 86)


donor site



 


Heavy chain
M A G :: G T R A G T


splice
(SEQ ID NO: 87)


donor site



 


Immunoglobulin
Y Y Y Y Y Y Y Y Y Y Y N C A G :: G


splice
(SEQ ID NO: 88)


acceptor site





Legend: Bases shown in bold are considered to be invariant within each consensus sequence. Splice sites are defined by the symbol “::”. Wobble bases are defined in Abbreviations (see Examples 9-11).













TABLE 9







Oligonucleotide primers used to


generate chimeric 158








Oligonu-



cleotide name
Sequence (5′→3′)





158vh
AAGCTTGCCGCCACCATGGACTCCAGGCTC


(SEQ ID



NO: 89)



 


158vhrev
GGGCCCTTGGTGGAGGCTGAGGAGACGGTGAC


(SEQ ID
TGAGG


NO: 90)



 


158vl
AAGCTTGCCGCCACCATGAAGTTGCCTGTTAGG


(SEQ ID



NO: 91)



 


158vlrev
GGATCCACTCACGTTTGATTTCCAGCTTGG


(SEQ ID
 


NO: 92)





Legend: Restriction sitesare underlined. Kozak sequences are in bold type.













TABLE 10







Oligonucleotide primers used for sequencing










Oligonucletotide name
Sequence (5′→3′)







1212 (17mer)
GTTTTCCCAGTCACGAC



(SEQ ID NO: 93)




 



1233 (24mer)
AGCGGATAACAATTTCACACAGGA



(SEQ ID NO: 94)




 



Hu-K2 (17mer)
CTCATCAGATGGCGGGA



(SEQ ID NO: 95)




 



BDSH61R
CGCTGCTGAGGGAGTAGAGTC



(SEQ ID NO: 96)

















TABLE 11





DNA sequence of 158 VK, primer MKV1 and the VK sequence derived using primers located 


within the V region















(SEQ ID NO: 97)









  1
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTG
158 VK


 







(SEQ ID NO: 98)









  1
.........VV...................
MKV1


 







(SEQ ID NO: 99)









  1
---------------------------------------------------------...A..G..........GG..............
*** VK


 


 91
CCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTAC
158 VK


 


 34
..........................................................................................
*** VK


 


181
CTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA
158 VK


 


124
..........................................................................................
*** VK


 


271
TCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCTCCG
158 VK


 


214
..........................................................................................
*** VK


 


361
ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTG
158 VK


 


304
.................................
*** VK





Legend: Residues identical to 158 VK are indicated by a dot. ***Sequencing using primers located within the V region.













TABLE 12





Chimeric VK leader sequence selection -


K5.1# leader selection for the chimeric VK
















158 VK
MKLPVRLLVLMFWIPASSS



(SEQ ID NO: 100)


 


K5.1#Protein
MKLPVRLLVLMFWIPASSS



(SEQ ID NO: 101)


 


K5.1#DNA
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGAT



GTTCTGGATTCCTGCTTCCAGCAGT



(SEQ ID NO: 102)
















TABLE 13







SignalP result 6 for K5.1# leader (SEQ ID NO: 103)


>Sequence length = 40












# Measure
Position
Value
Cut off signal peptide?















max.
C
20
0.970
0.32
YES


max.
Y
20
0.890
0.33
YES


max.
S
13
0.989
0.87
YES


mean 
S
1-19
0.954
0.48
YES



D
1-19
0.922
0.43
YES





# Highest probability for cleavage is between amino acid residue 19 and 20 (SSS-DV)













TABLE 14





Protein and DNA sequence of chimeric 158 VK construct



















    (SEQ ID NO: 104)   (SEQ ID NO: 105)




       81       162         243         324       405           421

















TABLE 15





DNA sequence of 158 VH, primer MHV5 and the sequence derived


using primers located within the V region

















1
ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTGATGTGCAGCTGGTGGAGTCT
158 VH



GGGGGAGGCTTA




(SEQ ID NO: 106)



1
---------------------------------------------------------..G...A.....A....A...
*** VH



............




(SEQ ID NO: 107)



1
..............................
MHV5



(SEQ ID NO: 108)



 


91
GTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTT
158 VH



CGTCAGGCTCCA



34
..............................................................................




............



 


181
GAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGGAGACACAGTGAAGGGCCGAT
158 VH



TCACCATCTCC



124
...............................................................................
*** VH



...........



 


271
AGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAA
158 VH



GAGAGGGGGGA



214
...............................................................................
*** VH



...........



 


361
TATTACTACGGTAGGAGTTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAA
158 VH



CAGCCCCA



304
...............................................................................
*** VH



........






Legend: Residues identical to 158 VH are indicated by a dot.


***Sequencing using primers located within the V region.













TABLE 16





Chimeric VH leader selection -


NL-1 VH leader sequence
















158 VH leader
MDSRLNLVFLVLILKGVQC



(SEQ ID NO: 109)


 


NL-1 protein
MDSRLNLVFLVLILKGVQC



(SEQ ID NO: 110)


 


NL-1 DNA
ATGGACTCCAGGCTCAATTTAGTTTTCCTT



GTCCTTATTTTAAAAGGTGTCCAGTGT



(SEQ ID NO: 111)
















TABLE 17







SignalP result 6 for NL-1 VH leader sequence (SEQ ID NO: 112)












# Measure
Position
Value
Cut off signal peptide?















max.
C
20
0.775
0.32
YES


max.
Y
20
0.795
0.33
YES


max.
S
13
0.953
0.87
YES


mean 
S
1-19
0.866
0.48
YES



D
1-19
0.830
0.43
YES





# Highest probability for cleavage is between amino acid residue 19 and 20 (VQC-DV) 19 and 20: VQC-DV













TABLE 18





Protein and DNA sequence of chimeric 158 VH



















    (SEQ ID NO: 113)   (SEQ ID NO: 114)




       81       162       243       324         405           461

















TABLE 19







Expression of chimeric 158 antibody in COS cells









Number of 
Expression Vector 
Antibody 


Co-transfections
Constructs Co-Transfected
Concentration (ng/ml)





2 pooled
pG1D200158 and pKN100158
 300


2 pooled
pG1D200158 and pKN100158
3700





Legend: Antibody concentration was measured by ELISA in 3-day cultures of transfected COS 7 cells. COS cells were co-transformed with 10 μg each of the heavy and light chain chimeric expression vectors pG1D200158 and pKN100158.













TABLE 20





Amino acid sequence of 158 VH and 158 VK
















VH
DVQLVESGGGLVQPGGSRKLSCAASGFTESSFGM



HWVRQAPEKGLEWVAYISSGSSTIYYGDTVKGRF



TISRDNPKNTLFLQMTSLRSEDTAMYYCAREGGY



YYGRSYYTMDYWGQGTSVTVES



(SEQ ID NO: 115)


 


VK
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGN



TYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS



GSGTDFTLKISRVEAEDLGIYYCFQGSHVPPTFGG



GTKLEIK



(SEQ ID NO: 116)
















TABLE 21







Best human VH framework VCI scores compared with 158 VH








Kabat Number6
2 24 26 27 28 29 30 37 39 45 47 48 49 67 69 71 73 78 91 93 94 103


Canonical Residue8
- 1  1  1  -  1  -  -  -  -  -  -  -  -  -  2  -  -  -  -  1  -


Vernier Residue7
* -  -  *  *  *  *  -  -  -  *  *  *  *  *  *  *  *  -  *  *  -


Interface Residue9
- -  -  -  -  -  -  I  I  I  I  -  -  -  -  -  -  -  I  I  -  I










Sequence name
FW score
VCI score
VCI Residues













158 VH
87
22
V A  G  F  T  F  S  V  Q  L  W  V  A  F  I  R  N  L  Y  A  R  W





(SEQ ID NO: 117)


 


38687
79
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB021520
77
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AJ556669
77
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


38672
77
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


38673
77
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


DQ322738
77
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB067108
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB021531
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB021532
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB063892
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB067237
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AB021507
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF471177
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF471184
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF062243
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF174030
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF466141
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AF466142
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AJ245279
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)


 


AJ579216
76
22
. .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 118)





Legend: Canonical residues are numbered in this table according to which CDR they are


associated with. FW score and VCI score are the number of residues in the FW or VCI


definition respectively, which are identical to their counterpart in 158. Residues identical to


those in 158 VH are indicated by a dot.













TABLE 22





Sequences of best VCI-scoring human VH, compared with 158 VH
















Kabat
          1         2         3           4         5            6         


Number6
-12345678901234567890123456789012345AB67890123456789012ABC34567890123456789


Canonical
                        1 11 1    1                    2   22


Vernier
  *                        ***                   ***                    * *


Interface
                         I I I                 I I


Kabat CDR
                     *******                        *******************





158 VH
-DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMH--WVRQAPEKGLEWVAYISS--GSSTIYYGDTVKGRFTI


AB021520
-EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AJ556669
-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKE--DGGEKFYVDSVKGRFTI


0Q322738
PLVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVAVIWY--DGSNKYYADSVKGRFTI 


AB067108
-EVQLVESGGGVVQPGGSLRLSCAASGFTFSNYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTI


A5021531
-QVQLVESGGGVVQPGRSLKLSCAASGFTFSSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTI


AB021532
-QVQLVESGGGVVQPGRSLKLSCAASGFTESSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTI


AB063892
-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AB067237
-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AB021507
-QVQLVESGGGVVQPGRSLKLSCAASGFTFSSYAMH--WVRQAPGKGLEWVAVISY--DGSNKYYADSVKGRFTI


AF471177
-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AF471184
-EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AF062243
CEVQLVESGGGLVQPGGSLRLSCSASGFTFSTYWMT--WVRQAPGKGLEWVANIKP--HGSEAYYVDSVKGRFTI


AF174030
CEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS--WVRQAPGKGLEWVANIKQ--DGSEKYYVDSVKGRFTI


AF466141
-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSAKGRFTI


AF466142
-QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSAKGRFTI


AJ245279
-QVQLVESGGGVVQPGGSLRLSCAASGFTFSSYGMH--WVRQAPGKGLEWVAVIWY--DGSNKYYADSVKGRFTI





Kabat
7         8            9        10                    11


Number6
0123456789012ABC345678901234567890ABCDEFGHILKlmnopqrstuv1234567890123


Canonical
 2                        1


Vernier
* *    *                 **                              *


Interface
                       I I I     I                       I


Kabat CDR
                            ******************************





158 VH
SRDNPKNTLFLQMTSLASEDTAMYYCAREGGYYYGRSYYT---------------MDYWGQGTSVTVSS



(SEQ ID NO: 119)


AB021520
SRDNAKNSLYLQMNSLRAEDTAVYYCARPDDSSGYYSAEY---------------FQHWGQGTLVTVSS



(SEQ ID NO: 120)


AJ556669
SRDNPKNSLFLQMNSLRAEDTAVYYCARERGHDFWSIYYTH--------------FDYWGQGALVTVSS



(SEQ ID NO: 121)


0Q322738
SRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSI----------------------FDYWGQGTLVTVSS



(SEQ ID NO: 122)


AB067108
SRDNSKNTLYLQMNSLRAEDTAVYYCARARDYYYYP-------------------MDVWGQGTTVTVSS



(SEQ ID NO: 123)


A5021531
SRDNSKNTLYLQMNSLRAEDTAVYYCARDQSWSRIAAAGTPPSL-----------FDPWGQGTLVTVSS



(SEQ ID NO: 124)


AB021532
SRDNSKNTLYLQMNSLRAEDTAVYYCARARNYYDSSGYS----------------FDYWGQGTLVTVSS



(SEQ ID NO: 125)


AB063892
SRDNAKNSLYLQMNSLRAEDTAVYYCARVRRGS----------------------GDSWGQGTLVTVSS



(SEQ ID NO: 126)


AB067237
SRDNAKNSLYLQMNSLRAEDTAVYYCAREQQLGPHNW------------------FDPWGQGTLVTVSS



(SEQ ID NO: 127)


AB021507
SRDNSKNTLYLQMNSLRAEDTAVYYCARDOETGTTFDYYYYG-------------MDVWGQGTTVTVSS



(SEQ ID NO: 128)


AF471177
SRDNAKNSLYLQMNSLRAEDTAVYYCARDPMTTVVKPSLAT--------------NDYWGQGTLVTVSS



(SEQ ID NO: 129)


AF471184
SRDNAKNSLYLQMNSLRAEDTAVYYCARDCVGALGA-------------------FDIWGQGTMVTVSS



(SEQ ID NO: 130)


AF062243
SRDNAKNSLFLQMSSLRAEDTAVYYCARANS------------------------LDVWGQGTTVTVSS



(SEQ ID NO: 131)


AF174030
SRDNAKNSLYLQMNSLRAEDTAVYYCARDGDIGDWW-------------------FDPWGQGTLVTVSS



(SEQ ID NO: 132)


AF466141
SRDNSKNTLFLQMNSLRAEDTAVYYCARDKGYYDYVWGSYRSNPKNDA-------FDIWGQGTMVTVSS



(SEQ ID NO: 133)


AF466142
SRDNSKNTLFLQMNSLRAEDTAVYYCARDKGYYDYVWGSYRSNPKNDA-------FDIWGQGTMVTVSS



(SEQ ID NO: 134)


AJ245279
SRDNSKNTLYLQMNSLRAEDTAVYYCARDRFF-----------------------FDNWGQGTLVTVSS



(SEQ ID NO: 135)
















TABLE 23





Alignment of 158 VH with the best VCI-scoring human VH





















          158 VH      AB021520     DQ322738      AB067108        AB021531     AB021532     AB063892     AB067237       AB021507       AF471177       AF471184      AF062243
               (SEQ ID NO: 136) (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139)   (SEQ ID NO: 140) (SEQ ID NO: 141) (SEQ ID NO: 142) (SEQ ID NO: 143) (SEQ ID NO: 144) (SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: 147)





Legend: Residues identical to 158 VH are represented by a dot. CDRs are grey-shaded.













TABLE 24





VH signal peptide selection - 5.5.1 V-gene alignment of 158 VH,


human AF062243 and human germline M99649 (VH3-07)

















DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYIS
158 VH
(SEQ ID NO: 148)


 


E................LR...S.......TYW.T......G.......N.K
AF062243
(SEQ ID NO: 149)


 


E................LR............YW.S......G.......N.K
M99649
(SEQ ID NO: 150)


 


SGSSTIYYGDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAR
158 VH
(SEQ ID NO: 148)


 


PHG.EA..V.S...........A..S.....S...A....V.....
AF062243
(SEQ ID NO: 149)


 


QDG.EK..V.S...........A..S.Y...N...A....V.....
M99699
(SEQ ID NO: 150)





Legend: V-gene residues identical to 158 VH are represented by a dot.













TABLE 25





Signal peptide of M99649 human germline VH gene

















DNA
ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGTCCAGTGT
(SEQ ID NO: 151)


 


protein
MELGLSWVFLVAILEGVQC
(SEQ ID NO: 152)
















TABLE 26







M99649 signal peptide cutting prediction (SEQ ID NO: 153)












# Measure
Position
Value
Cutoff signal peptide?















max.
C
20
0.909
0.32
YES


max.
Y
20
0.836
0.33
YES


max.
S
13
0.953
0.87
YES


mean
S
1-19
0.859
0.48
YES



D
1-19
0.848
0.43
YES





# Highest probability for cleavage is between amino acid residue 19-20: VQC-DV.













TABLE 27





Generation of 158RHA protein sequence (SEQ ID NOS: 154-163)
















Kabat
1         2         3         4           5         6


Number6
012345678901234567890123456789012345AB67890123456789012ABC34567890123456789





CDR
                     ===H1==              =====H2============


158 VH
-DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMH--WVRQAPEKGLEWVAYISS--GSSTIYYGDTVKGRFTI


 


158 CDR
SFGMH--(SEQ ID NO: 155) YISS--GSSTIYYGDTVKG (SEQ ID NO: 156)             EG


 


158RHA
-EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMH--WVRQAPGKGLEWVAYISS--GSSTIYYGDTVKGRFTI


 


AF062243 FW
-EVQLVESGGGLVQPGGSLRLSCSASGFTFS       WVRQAPGKGLEWVA RFTI (SEQ ID NO: 160)



(SEQ ID NO: 159) 


 


AF062243
CEVQLVESGGGLVQPGGSLRLSCSASGFTFSTYWMT--WVRQAPGKGLEWVANIKP--HGSEAYYVDSVKGRFTI





Kabat
7         8           9        10                   11


Number6
0123456789012ABC45678901234567890ABCDEFGHIJK1234567890123





CDR
                           =========H3========


158 VH
SRDNPKNTLFLQMTSLRSEDTAMYYCAREGGYYGRSYYT----MDYWGQGTSVTVSS (SEQ ID NO: 154)


 


158 CDR
GYYYGRSYYT----MDY (SEQ ID NO: 157)


 


158RHA
SRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYT----MDYWGQGTTVTVS (SEQ ID NO: 158)


 


AF062243 FW
SRDNAKNSLFLQMSSLRAEDTAVYYCAR (SEQ ID NO: 161)  WGQGTTVTVS (SEQ ID NO: 162)


 


AF062243
SRDNAKNSLFLQMSSLRAEDTAVYYCARANS-------------LDVWGQGTTVTVSS (SEQ ID NO: 163)
















TABLE 28







Generation of 158RHA DNA sequence-Generation of 158RHAss DNA sequence


 (SEQ ID NOS: 155-157, 159-162, and 164-172)









REGION
DNA
PROTEIN





VH3-07 leader
ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTA
MELGLSWVFLVAILEGVQC 



TTTTAGAAGGTGTCCAGTGT (SEQ ID NO: 164)
(SEQ ID NO: 165)


 


AF062243 FW1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTC
EVQLVESGGGLVQPGGSLRLS



CAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTC
CSASGFTFS



TGGATTCACCTTTAGT (SEQ ID NO: 166)
(SEQ ID NO: 159)


 

















(SEQ ID NO: 167)
(SEQ ID NO: 155)


 


AF062243 FW2
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCC
WVRQAPGKGLEWVA 



(SEQ ID NO: 168)
(SEQ ID NO: 160)


 

















(SEQ ID NO: 169)
(SEQ ID NO: 156)


 


AF062243 FW3
CGATTCACCATCTCCAGAGAGACAACGCCAAGAACTCACTG
RFTISRDNAKNSLFLQMSSLRA



TTTCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCCGTGTA
EDTAVYYCAR



TTATTGTGCGAGA (SEQ ID NO: 170)
(SEQ ID NO: 161)


 










  






(SEQ ID NO: 171)
(SEQ ID NO: 157)


 


AF062243 FW4
TGGGGCCAAGGGACCACGGTCACCGTCTCC
WGQGTTVTVS



(SEQ ID NO: 172)
(SEQ ID NO: 162)





Legend: Human VH3-07 leader and AF062243VH FWs intercalated with 158 VH CDRs (grey-shaded) to generate 158RHAss. (relate to Tables 27 and 28)













TABLE 29





DNA and protein sequence of 158RHAss

















(SEQ ID NO: 173)                (SEQ ID NO: 174)




    81                162          243          324            405         426





Legend: Splice donor sites predicted by Lasergene 6.0 GeneQuest analysis, together with their score, using the human_ds_2 matrix with a threshold of 4.2.













TABLE 30





Mutations in 158RHA removing splice sites in 158RHAss

















1
ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAGGGAGT
158RHA



(SEQ ID NO: 175)



1
............................................A..T..
158RHAss



(SEQ ID NO: 176)



 


51
CCAGTGCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG
158RHA


51
......T..G........................................
158RHAss


 


101
GGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTTAGTAGC
158RHA


101
..................................................
158RHAss


 


151
TTTGGAATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGT
158RHA


151
............................................G.....
158RHAss


 


201
GGCCTACATTAGTAGTGGCAGTAGTACCATCTACTATGGAGACACCGTGA
158RHA


201
.............................................A....
158RHAss


 


251
AGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTG
158RHA


251
..................................................
158RHAss


 


301
CAAATGAGCAGCCTGAGAGCCGAGGACACGGCCGTGTATTATTGTGCGAG
158RHA


301
..................................................
158RHAss


 


351
AGAGGGGGGATATTACTACGGAAGGAGTTACTATACTATGGACTACTGGG
158RHA


351
.....................T............................
158RHAss


 


401
GCCAAGGGACCACGGTCACCGTCTCC
158RHA


401
..........................
158RHAss





Legend 158RHA DNA sequence compared to 158RHAss (Table 5.7.2)


which contains predicted splice sites. Positions identical to


158RHA are identified as a dot.













TABLE 31





DNA and protein sequence of 158RHA

















(SEQ ID NO: 177)   (SEQ ID NO: 178)




 81               162             243             324             405         426 
















TABLE 32







Best VCI scores of human VK compared with 158 VK








Kabat Number6
2 4 35 36 38 44 46 47 48 49 64 66 68 69 71 87 98


Canonical Residue8
1           2  2      1


Vernier Residue7
****      *********       *


Interface Residue9
   IIII                  II










Sequence
Fw score
VCI score
VCI residues





158 VK
80
17
V M W  Y  Q  P  L  L  I  Y  G  G  G  T  F  Y  F





(SEQ ID NO: 179)


 


AB064054
71
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AB063934
70
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AB064105
70
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AY941999
70
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AX805665
69
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180


 


AB064104
69
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AY942057
69
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AB064055
68
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AX742874
68
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AY685343
67
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


AY685353
67
17
V . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 180)


 


DQ187506
70
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


DQ187679
70
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AY043107
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AJ388639
69
16
. . .  .  .  .  .  .  V  .  .  .  .  .   . .  .





(SEQ ID NO: 182)


 


AJ388646
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AJ388642
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


M74470
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


X72466
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


U95244
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AAA51016
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


X89054
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


DQ187505
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


DQ187683
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


DQ187691
69
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AX805669
68
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)


 


AF455562
68
16
I . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .





(SEQ ID NO: 181)





Legend: Canonical residues are numbered in this table according to which CDR they are


associated. FW score and VCI score are the number of residues in the FW or VCI defination


respectively, which are identical to their counterpart in 158. Residues identical to 158


VK are indicated by a dot.













TABLE 33





Sequences of best VCI-scoring human VK, compared with 158 VK

















         1         2               3         4         5


Kabat number6
123456789012345678901234567ABCDEF8901234567890123456789012345678





Canonical8
 1                      1   1     1   1              2


Vernier7
 * *                                    **         ****


Interface9
                                       F F F     F F


VCI
 1 *                    1   1     1   1F*F F     F ****


Kabat CDR
                        *****************              *******


158 VK
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHS-NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGV


AB064054
DVVMTQSPLSLPVTPGAPASISCRSSQSLLHT-NGVNFLDWYLQKPGQSPKLLIYLASHRASGV


AB063934
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AB064105 scFv
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AY941999 scFv
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AX805665 patent
DVVMTQSPLSLPVTPGEPASISCRSSQSIVHS-NGNTYLQWYLQKPGQSPQLLIYKVSNRLYGV


AB064104
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AY942057 scFv
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AB064055
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYSDWYLQKPGQSPQLLIYLGSSRASGV


AX742874
DVVMTQTPLSLSVTPGQPASISCRSSQSLLHS-DGMTYFSWYLQKPGQPPQLLIYEVSNRFSGV


AY685343
DVVMTQSPLSLAVTPGEPASISCRSSQSVVFT-NGKNYLDWYLQKPGQSPQLLIYLGSNRASGV


AY685353
DVVMTQSPLSLAVTPGEPASISCRSSQSVVFT-NGKNYLDWYLQKPGQSPQLLIYLGSNRASGV


DQ187506
DIVMTQTPLSLPVTPGEPASISCRSSQSLLES-HGYNYLDWYLQKPGQSPQLLIYLASNRPSGV


DQ187679
DIVMTQTPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AY043107
DIVMTOSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AJ388639
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYFAWYLQKPGQSPQLLVYLGSNRASGV


AJ388646
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AJ388642
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


M74470
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGFNYLHWYLQKPGQSPRLLIYLGSNRASGV


X72466
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHN-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


U95244
DIVMTOSPLSLPVTPGEPASISCRSSQSLLYS-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AAA51016
DivmTQSFLELPVTPGEPASISCRESQSLLKS-NGFNYLKWYLQKPGQSPRLLIYLGSNRASGV


X89054
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHS-NGYNYFDWYLQKPGQSPQLLIYLGSNRASGV


DQ187505
DIVMTOSPLSLPVTPGEPASISCRSSQSLLES-HGYNYLDWYLQKPGQSPQLLIYLASNRPSGV


DQ187683
DIVMTQTPLSLPVTPGEPASISCRSSQSLLHG-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


DQ187691
DIVMTQTPLSLPVTPGEPASISCRSSQSLLHG-NGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


AX805669
DIVMTQSPLSLPVTPGEPASISCRSSQSIVHS-NGNTYLQWYLQKPGQSPQLLIYKVSNRLYGV






          6         7         8         9              10


Kabat number6
         9012345678901234567890123456789012345ABCDEF678901234567





Canonical8
              2      1                  3    3       3


Vernier7
              * * ** *                                *


Interface9
                                     F F F          F F


VCI
              * * ** *               F F3F  33  *   F3F


Kabat CDR
                                       ***************





158 VK
         PDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVP------PTFGGGTKLEIK



         (SEQ ID NO: 183)


AB064054
         PDRFSGSGSGTDFTLRISRVEAEDVGIYYCMQGLQTP------FTFGPGTKLEIK



         (SEQ ID NO: 184)


AB063934
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------LTFGGGTKVEIK



         (SEQ ID NO: 185)


AB064105 scFv
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------YTFGQGTKLEIK



         (SEQ ID NO: 186)


AY941999 scFv
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------YTFGQGTKLEIK



         (SEQ ID NO: 187)


AX805665 patent
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP------WTFGQGTKVEIK



         (SEQ ID NO: 188)


AB064104
         PDGFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------HTFGQGTKLEIK



         (SEQ ID NO: 189)


AY942057 scFv
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQSP------PTFGRGTKVEIK



         (SEQ ID NO: 190)


AB064055
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------FTFGPGTKVDIK



         (SEQ ID NO: 191)


AX742874
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQNIQLP------WTFGQGTKVEIK



         (SEQ ID NO: 192)


AY685343
         PDRFSGSGSGTDFTLKISRVEADDVGVYYCMHAVQAP------WTFGQGTKVEIK



         (SEQ ID NO: 193)


AY685353
         PDRFSGSGSGTDFTLKISRVEADDVGVYYCMHAVQAP------WTFGQGTKVEIK



         (SEQ ID NO: 194)


DQ187506
         PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQNLQTP------YSFGQGTKLEIR



         (SEQ ID NO: 195)


DQ187679
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------HSFGQGTKLEIK



         (SEQ ID NO: 196)


AY043107
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------LTFGGGTKVEIK



         (SEQ ID NO: 197)


AJ388639
         PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQVLQTP------YTFGQGTKLEIS



         (SEQ ID NO: 198)


AJ388646
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------LTFGGGTKVEIK



         (SEQ ID NO: 199)


AJ388642
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------PTFGGGTKVEIK



         (SEQ ID NO: 200)


M74470
         PDRFSGSGSGTDFTLKISRVEADDVGIYYCMQALQSP------YTFGQGTKLEIK



         (SEQ ID NO: 201)


X72466
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQIP------LTFGGGTKVEIK



         (SEQ ID NO: 202)


U95244
         PDRFSGSGSGTDFTLKISRVEAEDVGDYYCMQALQSP------LTFGGGTKVEIK



         (SEQ ID NO: 203


AAA51016
         PDRFSGSGSGTUFTLKISRVEADDvGiyycmQALQSP------YTFGQGTKLEIK



         (SEQ ID NO: 204)


X89054
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------LTFGGGTKVEIK



         (SEQ ID NO: 205)


DQ187505
         PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQNLQTP------YSFGQGTKLEIR



         (SEQ ID NO: 206)


DQ187683
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------RTFGQGTKVEIK



         (SEQ ID NO: 207)


DQ187691
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP------RTFGQGTKVEIK



         (SEQ ID NO: 208)


AX805669
         PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP------WTFGQGTKVEIK



         (SEQ ID NO: 209)
















TABLE 34 





Alignment of 158 VK with the best VCI-scoring human VK
















Legend: CDR 1, 2 and 3 are grey-shaded.













TABLE 35





VK signal peptide selection-Alignment of 158 VK with human


AB064054 and human germline A19
















DVLMTQTPSLSPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSN
158 VK


(SEQ ID NO: 234)



..V...S......TP.AP..........LL.T..VNF.D...............LA.H
AB064054


(SEQ ID NO: 235)



.IV...S......TP.EP..........LL....YN..D..........Q....LG..
A19


(SEQ ID NO: 236)



 


RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK
158 VK


(SEQ ID NO: 234)



.A..................R........V.....M..LQT.F...P......N
AB064054


(SEQ ID NO: 235)



.A...........................V.V...M.ALQT.
A19


(SEQ ID NO: 236)
















TABLE 36







Signal peptide of human A19 


(VK2-28; X63397) germline VK











VK A19 leader sequence







DNA
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTG




CTAATGCTCTGGGTCTCTGGATCCAGTGGG




(SEQ ID NO: 237)



 



protein
MRLPAQLLGLLMLWVSGSSG




(SEQ ID NO: 238)

















TABLE 37







A19 signal peptide cutting prediction (SEQ ID NO: 239)


>Sequence length = 50












# Measure
Position
Value
Cutoff signal peptide?















max.
C
21
0.853
0.32
YES


max.
Y
21
0.831
0.33
YES


max.
S
13
0.990
0.87
YES


mean
S
1-20
0.932
0.48
YES



D
1-20
0.881
0.43
YES





# Most likely cleavage site between pos. 20 and 21: SSG-DV













TABLE 38





Generation of 158RKA Protein Sequence (SEQ ID NOS: 240-249)
















Kabat
                          1         2               3         4         5         


number 6
                 123456789012345678901234567ABCDEF89012345678901234567890123456789


CDR
                                        =======L1=======                 ==L2=== 


158 VK
                 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHS-NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVP


CDR
                        (SEQ ID NO: 241)RSSQSIVHS-NGNTYLE(SEQ ID NO: 242)KVSNRFS


158RKA
                 DVVMTQSPLSLPVTPGAPASISCRSSQSIVHS-NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVP


FW
(SEQ ID NO: 245) DVVMTQSPLSLPVTPGAPASISC(SEQ ID NO: 246) WYLQKPGQSPKLLIY      GVPD


AB064054
                 DVVMTQSPLSLPVTPGAPASISCRSSQSLLHT-NGVNFLDWYLQKPGQSPKLLIYLASHRASGVP













Kabat
6         7         8         9              10



number 6
012345678901234567890123456789012345ABCDEF678901234567



CDR
                             ======L3=======



158 VK
DRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVP------PTFGGGTKLEIK (SEQ ID NO: 240)



CDR
                             FQGSHVP------PT (SEQ ID NO: 243)     



158RKA
DRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVP------PTFGPGTKLEIK (SEQ ID NO: 244)



FW
RFSGSGSGTDFTLRISRVEAEDVGIYYC (SEQ ID NO: 247)FGPGTKLEIK (SEQ ID NO: 248)



AB064054
DRFSGSGSGTDFTLRISRVEAEDVGIYYCMQGLQTP------FTFGPGTKLEIK (SEQ ID NO: 249)






















TABLE 39







Generation of 158RKAss DNA Sequence


Intercalation of 158VK into AB064054 FW (SEQ ID NOS: 250-258)









Region
DNA
Protein





A19 leader
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTC
MRLPAQLLGLLMLWVSGSSG



TGGGTCTCTGGATCCAGTGGG(SEQ ID NO: 250)
(SEQ ID NO: 251)


 


AB064054 FW1
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCC
DVVMTQSPLSLPVTPGAPASISC



TGGAGCGCCGGCCTCCATCTCCTGC(SEQ ID NO: 252)
(SEQ ID NO: 245)


 

















(SEQ ID NO: 253)
(SEQ ID NO: 241)


 


AB064054 FW2
TGGTATCTGCAGAAGCCAGGGCAGTCTCCAAAGCTCCTGATCTAT
WYLQKPGQSPKLLIY



(SEQ ID NO: 254)
(SEQ ID NO: 246)


 

















(SEQ ID NO: 255)
(SEQ ID NO: 242)


 


AB064054 FW3
GGAGTCCCTGACAGGTTCAGTGGCAGTGGGTCAGGCACAGATTTTACAC
GVPDRFSGSGSGTDFTLRISRVEAE



TGAGAATCAGCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGC 
DVGIYYC 



(SEQ ID NO: 256)
(SEQ ID NO: 247)


 

















(SEQ ID NO: 257)
(SEQ ID NO: 243)


 


AB064054 FW4
TTCGGCCCTGGGACCAAATTGGAAATCAAA 
FGPGTKLEIK



(SEQ ID NO: 258)
(SEQ ID NO: 248)






















TABLE 40





158RKAss DNA sequence

















(SEQ ID NO: 259)   (SEQ ID NO: 260)




   81         162         243           324             396





Legend: Splice donor sites predicted by Lasergene 6.0 GeneQuest analysis, together with their score, using the human_ds_2 matrix with a threshold of 4.2.













TABLE 41





Mutations in 158RKA removing splice sites in 158RKA


















1
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTGGAAGCAGTGGG
158RKA
(SEQ ID NO: 261)


1
...................................................TC.......
158RKAss
(SEQ ID NO: 262)


 


61
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGCGCCGGCCTCC
158RKA



61
............................................................
158RKAss



 


121
ATCTCCTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAGTGG
158RKA



121
........................................................A...
158RKAss



 


181
TATCTTCAAAAGCCAGGGCAGTCTCCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTT
158RKA



181
.....G..G...................................................
158RKAss



 


241
TCTGGAGTCCCTGACAGGTTCAGTGGAAGTGGATCAGGCACAGATTTTACACTGAGAATC
158RKA



241
..........................C.....G...........................
158RKAss



 


301
AGCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCT
158RKA



301
............................................................
158RKAss



 


361
CCGACGTTCGGCCCTGGGACCAAATTGGAAATCAAA
158RKA



361
....................................
158RKAss





Legend: 158RKA DNA sequence compared to 158RKAss (Table 5.13.2) which contains


predicted splice sites. Residues identical to 158RKA are identified by a dot.













TABLE 42





DNA and protein sequence of 158RKA

















(SEQ ID NO: 263)   (SEQ ID NO: 264)




 81         162         243         324          396    
















TABLE 43





Further version of humanized 158VHA (158RHB,158RHC,158RHD)



























TABLE 44





Further version of humanized 158VKA (158RKB,158RKC)

## Claims

1. A method of measuring Aβ protofibrils in a mammal, said method comprising the steps of:
(a) providing a labeled antibody or fragment thereof, being selective and having high affinity for human Aβ protofibrils, and wherein the labeled antibody or fragment thereof in its six CDR regions comprises:












(SEQ ID NO: 1)



VH-CDR1
SFGMH;



 




(SEQ ID NO: 2)



VH-CDR2
YISSGSSTIYYGDTVKG;



 




(SEQ ID NO: 3)



VH-CDR3
EGGYYYGRSYYTMDY;



 




(SEQ ID NO: 4)



VL-CDR1
RSSQSIVHSNGNTYLE;



 




(SEQ ID NO: 5)



VL-CDR2
KVSNRFS;




and



 




(SEQ ID NO: 6)



VL-CDR3
FQGSHVPPT,




wherein






said antibody or fragment thereof is labeled with an agent that generates a measurable signal;
(b) administering the labeled antibody or fragment thereof to a mammal comprising or suspected of comprising Aβ protofibrils; and
(c) measuring the concentration of a complex formed between said Aβ protofibril and said antibody or fragment thereof by measuring the signal generated by the agent.

2. The method of claim 1, wherein the agent is labeled with a radioactive ligand.

3. The method of claim 2, wherein the radioactive ligand is selected from the group I131, C14, H3, or Gallium68.

4. The method of any one of claims 1-3, wherein said measuring is used in the diagnosis of Alzheimer's disease or Down's syndrome.